# Patent

## Title
Infarct area perfusion-improving compositions and methods of vascular injury repair

## Abstract

The described invention provides pharmaceutical compositions for treating an infarct area injury and methods of treating or repairing the infarct area injury in a revascularized subject in the aftermath of an acute myocardial infarction resulting from a natural disease process by administering to the subject parenterally through a catheter a sterile pharmaceutical composition containing a therapeutically effective amount of a nonexpanded sterile isolated chemotactic hematopoietic stem cell product as a first therapeutic agent and optionally a therapeutically effective amount of at least one compatible second therapeutic agent. The infarct area-improving amount of the sterile isolated chemotactic hematopoietic stem cell product comprises an enriched population of isolated autologous CD34+ cells containing a subpopulation of potent cells expressing CXCR-4 and having CXCR-4-mediated chemotactic activity such that the enriched population of isolated autologous CD34+ hematopoietic stem cells provides at least 0.5×106 potent CD34+ cells expressing CXCR-4 and having CXCR-4 mediated chemotactic activity.

## Background
CROSS REFERENCE TO RELATED APPLICATIONS
This application claims the benefit of the filing date of U.S. application Ser. No. 11/552,396 (filed Oct. 24, 2006), U.S. Provisional Application No. 61/119,552, filed Dec. 3, 2008, and U.S. Provisional Application 61/169,850, filed Apr. 16, 2009. The contents of each of these applications is incorporated by reference herein in its entirety.


FIELD OF THE INVENTION
The described invention relates to infarct area perfusion-improving compositions comprising a chemotactic hematopoietic stem cell product and methods of use thereof in repairing an infarct area injury in the aftermath of an acute myocardial infarction resulting from a natural disease process in a revascularized subject.
BACKGROUND OF THE INVENTION
The Cardiac Cycle
The term “diastole” refers to the normal postsystolic dilation of the heart cavities during which they full with blood. The term “systole” refers to contraction of the heart, especially of the ventricles, by which the blood is driven through the aorta and pulmonary artery to traverse the systemic and pulmonary circulations, respectively.
The term “cardiac cycle” is used to refer to all or any of the mechanical events related to the coronary blood flow or blood pressure that occurs from the beginning of one heartbeat to the beginning of the next. Blood pressure increases and decreases throughout the cardiac cycle. The frequency of the cardiac cycle is the heart rate. Every single ‘beat’ of the heart involves five major stages: (1)“Late diastole,” which is when the semilunar valves close, the atrioventricular (Av) valves open and the whole heart is relaxed; (2) “Atrial systole,” which is when the myocardium of the left and right atria are contracting, AV valves open and blood flows from atrium to ventricle; (3) “Isovolumic ventricular contraction,” which is when the ventricles begin to contract, AV and semilunar valves close, and there is no change in volume; (4) “ventricular ejection,” which is when the ventricles are empty but still contracting and the semilunar valves are open; and (5) “Isovolumic ventricular relaxation,” when pressure decreases, no blood is entering the ventricles, the ventricles stop contracting and begin to relax, and the semilunar valves are shut because blood in the aorta is pushing them shut. The cardiac cycle is coordinated by a series of electrical impulses that are produced by specialized heart cells found within the sino-atrial node and the atrioventricular node.
Coronary Blood Flow
The flow of blood through the coronary arteries is pulsatile, with characteristic phasic systolic and diastolic flow components. Systolic flow, which relates to the contraction or pumping phase of the heart cycle, has rapid, brief, retrograde responses. Diastolic flow, which relates to the relaxation or filling phase of the heart cycle, occurs during the relaxation phase after myocardial contraction, with an abrupt increase above systolic levels and a gradual decline parallel with that of aortic diastolic pressures. Intramural coronary blood volume changes during each heartbeat, with the myocardium accommodating the volume change brought about by muscular contraction. Coronary venous flow is out of phase with coronary arterial flow, occurring predominantly in systole and nearly absent during diastole.
For each heartbeat, blood pressure varies between systolic and diastolic pressures. The term “systolic pressure” refers to the peak pressure in the arteries, which occurs near the end of the cardiac cycle when the ventricles are contracting. The term “diastolic pressure” refers to the minimum pressure in the arteries, which occurs near the beginning of the cardiac cycle when the ventricles are filled with blood.
Coronary blood flow not only is phasic but also varies with the type of vessel and location in the myocardium. Coronary arterioles appear to have specialized regulatory elements along their length that operate “in series” in an integrated manner. A system of multiple functional “valves” permits fine control of the coronary circulation. The smallest arterioles dilate during metabolic stress, resulting in reduced microvascular resistance and increased myocardial perfusion. Stenosis or narrowing of a blood vessel produces resistance to blood flow related directly to the morphologic features of the stenosis. As the upstream ateriolar pressure decreases due to a fall in distending pressure across the stenosis, myogenic dilation of slightly larger arterioles upstream occurs and causes an additional decrease in resistance. Increased flow in the largest arterioles augments shear stress and triggers flow-mediated dilation, further reducing the resistance of this network.
The arterial and venous pulsatile flow characteristics of the heart are dependent on intramyocardial compliance. The term “compliance” refers to a measure of the tendency of a hollow organ to resist recoil toward its original dimensions upon removal of a distending or compressing force. The higher the compliance the more elastic the material. Compliance is calculated using the following equation, where ΔV is the change in volume, and ΔP is the change in pressure:




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  =
  
    
      Δ
      ⁢
      
        
      
      ⁢
      V
    
    
      Δ
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The capacity of the heart as a reservoir is controlled by resistance arterioles to coronary vascular inflow. Outlet resistance is related to intramural cardiac veins. The intramyocardial capillary resistance influences both arterial and venous responses but predominantly acts in concert with outlet resistance.
Approximately 75% of total coronary resistance occurs in the arterial system, which comprises conductance (R1), prearteriolar (R2) and arteriolar and intramyocardial capillary vessels (R3). Normal epicardial coronary arteries in humans typically are 0.3 to 5 mm in diameter, and do not offer appreciable resistance to blood flow. Normally, large epicardial vessel resistance (R1) is trivial until atherosclerotic obstructions compromise the lumen. Precapillary arterioles (R2), 100 to 500 μm in size) are resistive vessels connecting epicardial to myocardial capillaries and are the principal controllers of coronary blood flow. They contribute approximately 25% to 35% of total coronary resistance. Distal precapillary arteriolar vessels (<100 μm in diameter), the main site of metabolic regulation of coronary blood flow, are responsible for 40-50% of coronary flow resistance. The dense network of about 4000 capillaries per square millimeter ensures that each myocyte is adjacent to a capillary. Capillaries are not uniformly patent (meaning open; affording free passage), because precapillary sphincters regulate flow according to the needs of the myocardium.
Several conditions, such as left ventricular hypertrophy, myocardial ischemia, or diabetes, can impair the microcirculatory resistance (R3), blunting the maximal absolute increase in coronary flow in times of increased oxygen demand.
Ischemia
The myocardium depends almost entirely on aerobic metabolism, since oxygen stores in the heart are meager. Myocardial oxygen supply rises and falls in response to the oxygen (energy) demands of the myocardium. The term “autoregulation” refers to the ability to maintain myocardial perfusion at constant levels in the face of changing driving forces. Autoregulation maintains coronary perfusion at relatively constant levels over a wide range of mean aortic pressure. When aortic pressure exceeds its upper or lower limits, coronary blood flow precipitously declines or increases proportionately.
The heart needs to be supplied with a sufficient quantity of oxygen to prevent underperfusion. When reduced perfusion pressure distal to stenoses is not compensated by autoregulatory dilation of the resistance vessels, ischemia, meaning a lack of blood supply and oxygen, occurs. Because the zone least supplied generally is the farthest out, ischemia generally appears in areas farthest away from the blood supply.
After total or near-total occlusion of a coronary artery, myocardial perfusion occurs by way of collaterals, meaning vascular channels that interconnect epicardial arteries. Collateral channels may form acutely or may preexist in an under-developed state before the appearance of coronary artery disease. Preexisting collaterals are thin-walled structures ranging in diameter from 20 μm to 200 μm, with a variable density amoung different species. Preexisting collaterals normally are closed and nonfunctional, because no pressure gradient exists to drive flow between the arteries they connect. After coronary occlusion, the distal pressure drops precipitously and prexisting collaterals open virtually instantly.
The term “myocardial ischemia” refers to a decrease in blood supply and oxygen to the cells of the myocardium. The development of myocardial ischemia has been attributed to two mechanisms: (1) increased myocardial oxygen demand, and (2) decreased myocardial perfusion and oxygen delivery. (Willerson, J. T. et al, JACC 8(1): 245-50 (1986)). Myocardial ischemia generally appears first and is more extensive in the subendocardial region, since these deeper myocardial layers are farthest from the blood supply, with greater need for oxygen.
Transient ischemia, hibernating myocardium, and myocardial infarction are clinically different conditions.
Transient Ischemia. The term “transient ischemia” as used herein refers to a reversible (meaning that the myocytes survive the insult) narrowing of a coronary artery at rest or with exercise where there is no thrombus or plaque rupture but where blood supply cannot be met. Every time the heart's oxygen demand increases, an imbalance between oxygen demand and supply is created. Transient ischemia produces a cascade of events beginning with metabolic and biochemical alterations leading to impaired ventricular relaxation and diastrolic dysfunction, impaired systolic function, and electrocardiographic abnormalities with ST segment alterations, followed by increased end-diastolic pressure with left ventricular dyssynchrony, hypokinesis, akinesis, and dyskinesis, and lastly painful symptoms of angina. Even though ischemic myocytes experience physiological and metabolic changes within seconds of the cessation of coronary flow, resulting in T wave and sometimes ST segment abnormalities (but without serum enzyme elevation), no cell death results from the ischemia. Kloner, R. A. and Jennings, R B, Circulation 104: 2981-89 (2001). Once blood flow is re-established, a complete recovery of myocyte contractile function takes place.
Although angina pectoris (chest pain) may be a symptom of transient ischemia, by and large transient ischemia is silent (meaning ST-segment depression of at least 1 mm is present without associated symptoms, e.g., chest pain) in 79% of subjects. In most patients with stable angina, for example, physical effort or emotion, with a resultant increase in heart rate, blood pressure, or contractile state, or any combination thereof, increases myocardial oxygen demand without an adequate delivery in oxygen delivery through tightly narrowed (stenosed) coronary arteries. More than 40% of patients with stable angina treated with one or more antianginal drugs have frequent episodes of silent ischemia, which has been shown to predict a higher risk of coronary events and cardiac death. Deedwania, P C, Carbajal, E V, Arch. Intern. Med. 150: 2373-2382 (1991).
Chronic Myocardial Ischemia. The term “chronic myocardial ischemia (CMI)” as used herein refers to a prolonged subacute or chronic state of myocardial ischemia due to narrowing of a coronary blood vessel in which the myocardium “hibernates”, meaning that the myocardium downregulates or reduces its contractility, and hence its myocardial oxygen demand, to match reduced perfusion, thereby preserving cellular viability and evading apoptosis. The underlying mechanism by which the myocardium does so is poorly understood. This hibernating myocardium is capable of returning to normal or near-normal function on restoration of an adequate blood supply. Once coronary blood flow has been restored to normal or near normal and ischemia is resolved, however, the hibernating myocardium still does not contract. This flow-function mismatch resulting in a slow return of cardiac function after resolution of ischemia has been called stunning. The length of time for function to return is quite variable, ranging from days to months, and is dependent on a number of parameters, including the duration of the original ischemic insult, the severity of ischemia during the original insult, and the adequacy of the return of the arterial flowA number of studies have provided evidence for inflammation in hibernating myocardium. Heusch, G. et al., Am. J. Physiol. Heart Circ. Physiol. 288: 984-99 (2005). A study conducted in a porcine model of myocardial hibernation in which the mean rest LAD coronary blood flow was reduced to about 60% of baseline for a period of 24 hours to four weeks, detected apoptotic myocytes in all experimental pigs in the hibernating regions supplied by the stenotic LAD, suggesting that functional downregulation may not be adequate to prevent gradual, ongoing myocyte death through apoptosis in hibernating myocardium. Chen, C, et al., J. Am. Coll. Cardiol. 30: 1407-12 (1997). A biopsy study of human patients undergoing coronary artery bypass surgery, likewise recognized myocyte apoptosis, which negatively influences left ventricle functional recovery, as an important phenomenon in hibernating myocardium. Angelini, A., et al, Eur. J. Heart Failure 9(4): 377-83 (2006).
Acute Myocardial Infarction (AMI). Another type of insult occurs during AMI. AMI is an abrupt change in the lumen of a coronary blood vessel which results in ischemic infarction, meaning that it continues until heart muscle dies. On gross inspection, myocardial infarction can be divided into two major types: transmural infacts, in which the myocardial necrosis involves the full or nearly full thickness of the ventricular wall, and subendocardial (nontransmural) infarcts, in which the myocardial necrosis involves the subendocardium, the intramural myocardium, or both, without extending all the way through the ventricular wall to the epicardium. There often is total occlusion of the vessel with ST segment elevation because of thrombus formation within the lumen as a result of plaque rupture. The prolonged ischemic insult results in apoptotic and necrotic cardiomyocyte cell death. See Kajstura, J., et al., Lab Invest. 74: 86-107 (1996). Necrosis compromises the integrity of the sarcolemmal membrane and intracellular macromolecules such that serum cardiac markers, such as cardiac-specific troponins and enzymes, such as serum creatine kinase (CK), are released. In addition, the patient may have electrocardiogram (ECG) changes because of full thickness damage to the muscle. An ST-Elevation Myocardial Infarction (STEMI) is a larger injury than a non-ST-elevation myocardial infarction. ST-segment elevation and Q waves on the ECG, two features highly indicative of myocardial infarction, are seen in only about half of myocardial infarction cases on presentation.
Acute myocardial infarction remains common with a reported annual incidence of 1.1 million cases in the United States alone (Antman, E. M., Braunwald, E., Acute Myocardial Infarction, in Principles of Internal Medicine, 15th Ed., Braunwald, E. et al., Eds., New York: McGraw-Hill (2001)). Preclinical and clinical data demonstrate that following a myocardial infarction, the acute loss of myocardial muscle cells and the accompanying peri-infarct zone hypo-perfusion result in a cascade of events causing an immediate diminution of cardiac function, with the potential for long term persistence. The extent of myocardial cell loss is dependent on the duration of coronary artery occlusion, existing collateral coronary circulation and the condition of the cardiac microvasculature. Paul et al., Am. Heart J. 131: 710-15 (1996); Pfeffer, M. A., Braunwald, E., Circulation 81: 1161-72 (1990); Sheilban, I. et. al., J. Am. Coll. Cardiol. 38: 464-71 (2001); Braunwald E., Bristow, M. R., Circulation 102: IV-14-23 (2000); Rich et al., Am. J. Med. 92:7-13 (1992); Ren et al., J. Histochem. Cytochem. 49: 71-79 (2002); Hirai, T. et al., Circulation 79: 791-96 (1989); Ejiri, M. et al., J. Cardiology 20: 31-37 (1990). Because myocardial cells have virtually no ability to regenerate, myocardial infarction leads to permanent cardiac dysfunction due to contractile-muscle cell loss and replacement with nonfunctioning fibrotic scarring. Frangogiannis, N. G., et al., Cardiovascular Res. 53(1): 31-47 (2002). Moreover, compensatory hypertrophy of viable cardiac muscle leads to microvascular insufficiency that results in further demise in cardiac function by causing myocardial muscle hibernation and apoptosis of hypertrophied myocytes in the peri-infarct zone.
Among survivors of myocardial infarction, residual cardiac function is influenced by the extent of ventricular remodeling (meaning changes in size, shape, and function, typically a progressive decline in function, of the heart after injury). Alterations in ventricular topography (meaning the shape, configuration, or morphology of a ventricle) occur in both infarcted and healthy cardiac tissue after myocardial infarction. Pfeffer, M. A., Braunwald, E., Circulation 81: 1161-72 (1990). Ventricular dilatation (meaning a stretching, enlarging or spreading out of the ventricle) causes a decrease in global cardiac function and is affected by the infarct size, infarct healing and ventricular wall stresses. Recent efforts to minimize remodeling have been successful by limiting infarct size through rapid reperfusion (meaning restoration of blood flow) using thromobolytic agents and mechanical interventions, including, but not limited to, placement of a stent, along with reducing ventricular wall stresses by judicious use of pre-load therapies and proper after-load management. Id. Regardless of these interventions, a substantial percentage of patients experience clinically relevant and long-term cardiac dysfunction after myocardial infarction. Sheiban, I. et al., J. Am. Coll. Cardiol. 38: 464-71 (2001). Despite revascularization of the infarct related artery circulation and appropriate medical management to minimize ventricular wall stresses, a significant percentage of these patients experience ventricular remodeling, permanent cardiac dysfunction, and progressive deterioration of cardiac function, and consequently remain at an increased lifetime risk of experiencing adverse cardiac events, including death. Paul et al., Am. Heart J. 131: 710-15 (1996); Pfeffer, M. A., Braunwald, E., Circulation 81: 1161-72 (1990).
At the cellular level, immediately following a myocardial infarction, transient generalized cardiac dysfunction uniformly occurs. In the setting of a brief (i.e., lasting three minutes to five minutes) coronary artery occlusion, energy metabolism is impaired, leading to demonstrable cardiac muscle dysfunction that can persist for up to 48 hours despite immediate reperfusion. This so-called “stunned myocardium phenomenon” occurs subsequent to or after reperfusion and is thought to be a result of reactive oxygen species. The process is transient and is not associated with an inflammatory response. Frangogiannis, N. G., et al., Cardiovascular Res. 53(1): 31-47 (2002). After successful revascularization, significant recovery from stunning occurs within three to four days, although complete recovery may take much longer. Boli, R., Prog. Cardiovascular Disease 40(6): 477-515 (1998); Sakata, K. et al., Ann. Nucleic Med. 8: 153-57 (1994); Wollert, K. C. et al., Lancet 364: 141-48 (2004).
Coronary artery occlusion of more significant duration, i.e., lasting more than five minutes, leads to myocardial ischemia and is associated with a significant inflammatory response that begins immediately after reperfusion and can last for up to several weeks. Frangogiannis, N. G., et al., Cardiovascular Res. 53(1): 31-47 (2002); Frangogiannis, N. G. et al., Circulation 98: 687-798 (1998).
The inflammatory process following reperfusion is complex. Initially it contributes to myocardial damage but later leads to healing and scar formation. This complex process appears to occur in two phases. In the first so-called “hot” phase (within the first five days), reactive oxygen species (in the ischemic myocardial tissue) and complement activation generate a signal chemotactic for leukocytes (chemotaxis is the directed motion of a motile cell, organism or part towards environmental conditions it deems attractive and/or away from surroundings it finds repellent) and initiate a cytokine cascade. Lefer, D. J., Granger, D. N., Am. J. Med. 4:315-23 (2000); Frangogiannis, N. G., et al., Circulation 7:699-710 (1998). Mast cell degranulation, tumor necrosis factor alpha (TNFα) release, and increased interleukin-6 (IL-6), intercellular adhesion molecule 1 (“ICAM-1” or CD-54, a receptor typically expressed on endothelial cells and cells of the immune system), selectin (L, E and P) and integrin (CD11a, CD11b and CD18) expression all appear to contribute to neutrophil accumulation and degranulation in ischemic myocardium. Frangogiannis, N. G. et al., Circulation 7: 699-710 (1998), Kurrelmeyer, K. M, et al., Proc. Nat'l Acad. Sci. 10: 5456-61 (2000); Lasky, L. A., Science 258: 964-69 (1992); Ma, X. L., et al., Circulation 88(2): 649-58 (1993); Simpson, P. J. et al., J. Clin. Invest. 2: 624-29 (1998). Neutrophils contribute significantly to myocardial cell damage and death through microvascular obstruction and activation of neutrophil respiratory burst pathways after ligand-specific adhesion to cardiac myocytes. Entman, M. L., et al., J. Clin. Invest. 4: 1335-45 (1992). During the “hot” phase, angiogenesis is inhibited due to the release of angiostatic substances, including interferon gamma-inducible protein (IP 10). Frangogiannis, N. G., et al., FASEB J. 15: 1428-30 (2001).
In the second phase, the cardiac repair process begins (about day 6 to about day 14), which eventually leads to scar formation (about day 14 to about day 21) and subsequent ventricular remodeling (about day 21 to about day 90). Soon after reperfusion, monocytes infiltrate the infarcted myocardium. Attracted by complement (C5a), transforming growth factor B1 (“TGF-B1”) and monocyte chemotactic protein 1 (“MCP-1”), monocytes differentiate into macrophages that initiate the healing process by scavenging dead tissue, regulating extracellular matrix metabolism, and inducing fibroblast proliferation. Birdshall, H. H., et al., Circulation 3: 684-92 (1997). Secretion of interleukin 10 (IL-10) by infiltrating lymphocytes also promotes healing by down-regulating inflammatory cytokines and influencing tissue remodeling. Frangogiannis, N. G. et al., J. Immunol. 5:2798-2808 (2000). Mast cells also appear to be involved in the later stages of myocardial repair by participating in the formation of fibrotic scar tissue. Stem Cell Factor (SCF) is a potent attractor of mast cells. SCF mRNA has been shown to be up-regulated in ischemic myocardial segments in a canine model of myocardial infarction and thus may contribute to mast cell accumulation at ischemic myocardial sites. Franigogiannis, N. G. et al., Circulation 98: 687-798 (1998). Mast cell products (including TGF-B, basic fibroblast growth factor (bFGF), vascular endothelial growth factor (VEGF) and gelatinases A and B) induce fibroblast proliferation, influence extracellular matrix metabolism, and induce angiogenesis. Fang, K. C., et al., J. Immunol. 162: 5528-35 (1999); Takeshi, S., et al., Cardiology 93: 168-74 (2000).
Following a myocardial infarction, neoangiogenesis occurs after the “hot” phase of the inflammatory process subsides (about day 5) coincident with rising levels of VEGF (VEGF peaks at about day 7 and gradually subsides to baseline at about day 14 to about day 21). During this phase of the healing process, endothelial precursor cells (EPCs) are mobilized and recruited to the infarct site. Shinitani, S., et al., Circulation 103: 2776-79 (2001). Without being limited by theory, it has been suggested that the chemokine stromal cell derived factor-1 (SDF-1), which is the ligand for the CXCR-4 chemokine receptor expressed by CD34+ cells, also plays a role in homing of cells to areas of ischemic damage. Ceredini, D. J., et al., Nature Medicine 10: 858-63 (2004); Askari, A., et al., Lancet 362: 697-703 (2003); Yamaguchi, J. et al., Circulation 107: 1322-34 (2003). While it is known that SDF-1 plays a role in hematopoiesis and is involved in migration, homing and survival of hematopoietic progenitors, and while SDF-1 has been implicated in ischemic neovascularization in vivo by augmenting EPC recruitment to ischemic sites (Yamaguchi et al. Circulation 107:1322-1328 (2003), SDF-1's role in neoangiogenesis is not certain. There is suggestive evidence implicating SDF-1. For example, SDF-1 gene expression is upregulated during hypoxia, a deficiency of oxygen in the tissues, by hypoxia inducible factor-1 (HIF-1). Furthermore, CD34+ cells are capable of homing to areas of ischemia, rich in SDF-1, including infarcted myocardium. Askari et al., Lancet 362: 697-703 (2003). Moreover, virtually all cells that express VEGF-2 coexpress CD34 and CXCR-4, but only about 1% to about 2% of CD34+CXCR-4+ cells co-express VEGF-2.
The Peri-Infarct Border Zone
The zone of dysfunctional myocardium produced by coronary artery occlusion extends beyond the infarct region to include a variable boundary of adjacent normal appearing tissue. (Hu, Q., et al., Am. J. Physiol. Heart Circ. Physiol. 291: H648-657 (2006)). This ischemic, but viable, pen-infarct zone of tissue separates the central zone of progressive necrosis from surrounding normal myocardium. The peri-infarct zone does not correlate with enzymatic parameters of infarct size and is substantially larger in small infarcts. Stork, A., et al., European Radiol. 16(10): 2350-57 (2006).
Ischemia due to edema and compression of the blood vessels in the border zone may be very important to outcome. It is known, for example, that after an AMI, transient ischemia occurs in the border zones and that percutaneous coronary interventions, which open up the infarct-related artery, can adversely affect the health of the pen-infarct border zones. It has been suggested that intermediate levels of mean blood flow can exist as the result of admixture of peninsulas of ischemic tissue intermingled with regions of normally perfused myocardium at the border of an infarct. (Hu, Q., et al., Am. J. Physiol. Heart Circ Physiol. 291: H648-657 (2006)). However, the boundary of the intermingled coronary microvessels, which in dogs is no more than 3 mm in width, cannot explain the relatively broad region of dysfunctional myocardium surrounding an infarct. Murdock, R H, Jr., et al., Cir. Res. 52: 451-59 (1983); Buda, A J, et al., J. Am. Coll. Cariol. 8: 150-58 (1986). Progressive dysfunction of this pen-infarct myocardium over time may contribute to the transition from compensated remodeling to progressive heart failure after an AMI.
To date, no ideal therapy exists for preventing the long term adverse consequences of vascular insufficiency, particularly the significant vascular insufficiency after a myocardial infarction. While large vessel revascularization (meaning the successful placement of a stent) seems promising, studies to date have shown such applications to be insufficient in addressing increased demands posed by compensatory myocardial hypertrophy. As a result, infarct extension and fibrous replacement commonly occur, regardless of large vessel revascularization, appropriate medical management of ventricular wall stresses, and potential natural, albeit suboptimal, CD34+ cell-mediated neoangiogenesis (one of the theories relating to the underlying cause of myocardial infarction is that the ability to mobilize these cells may be biologically limited).
Intense interest has developed in evaluating the ability of endothelial and myocardial precursor cells to limit damage to the myocardium after infarction and to limit or prevent ventricular remodeling. Significant preclinical data and some clinical data demonstrate the safety and potential of cell therapy using a variety of cell precursors (particularly hematopoietic cells) to contribute to neoangiogenesis, limited cardiac myogenesis (principally by fusion), and muscle preservation in the myocardial infarct zone. See, e.g., Jackson, et al., J. Clin. Invest. 107: 1395-1402 (2001); Edelberg, J. M., et al., Cir. Res. 90: e89-e93 (2002); Schichinger, V. et al., New Engl. J. Med. 355 (12): 1210-21 (2006) (using bone marrow-derived progenitor cells); Assmus, B. et al., New Engl. J. Med. 355 (12) 1222-32 (2006) (using bone marrow-derived progenitor cells), but see Lunde, K. et al., New Eng. J. Med. 355 (12): 1199-209 (2006) (using bone marrow-derived progenitor cells). It is not known under what circumstances or the extent to which left ventriclar remodeling is reversible.
Bone marrow consists of a variety of precursor and mature cell types, including hematopoietic cells (the precursors of mature blood cells) and stromal cells (the precursors of a broad spectrum of connective tissue cells), both of which appear to be capable of differentiating into other cell types. Wang, J. S. et al., J. Thorac. Cardiovasc. Surg. 122; 699-705 (2001); Tomita, S. et al., Circulation 100 (Suppl. II): 247-256 (1999); Saito, T. et al., Tissue Eng. 1: 327-43 (1995). Unmodified (i.e., not fractionated) marrow or blood-derived cells have been used in several clinical studies, for example, Hamano, K. et al., Japan Cir. J. 65: 845-47 (2001); Strauer, B. E., et al., Circulation 106: 1913-18 (2002); Assmus, et al., Circulation 106: 3009-3017 (2002); Dobert, N. et al., Eur. J. Nuel. Med. Mol. Imaging, 8: 1146-51 (2004); Wollert, K. C. et al., Lancet 364: 141-48 (2004). Since the mononuclear fraction of bone marrow contains stromal cells, hematopoietic precursors, and endothelial precursors, the relative contribution of each of these populations to the observed effects, if any, remains unknown.
CD34 is a hematopoietic stem cell antigen selectively expressed on hematopoietic stem and progenitor cells derived from human bone marrow, blood and fetal liver. Yin et al., Blood 90: 5002-5012 (1997); Miaglia, S. et al., Blood 90: 5013-21 (1997). Cells that express CD34 are termed CD34+. Stromal cells do not express CD34 and are therefore termed CD34−. CD34+ cells isolated from human blood may be capable of differentiating into cardiomyocytes, endothelial cells, and smooth muscle cells in vivo. See Yeh, et al., Circulation 108: 2070-73 (2003). CD34+ cells represent approximately 1% of bone marrow derived nucleated cells; CD34 antigen also is expressed by immature endothelial cell precursors, while mature endothelial cells do not express CD34. Peichev, M. et al., Blood 95: 952-58 (2000). In vitro, CD34+ cells derived from adult bone marrow give rise to a majority of the granulocyte/macrophage progenitor cells (CFU-GM), some colony-forming units-mixed (CFU-Mix) and a minor population of primitive erythroid progenitor cells (burst forming units, erythrocytes or BFU-E). Yeh, et al., Circulation 108: 2070-73 (2003). CD34+ cells also may have the potential to differentiate into, or to contribute to, the development of new myocardial muscle, albeit at low frequency.
Techniques have been developed using immunomagnetic bead separation to isolate a highly purified and viable population of CD34+ cells from bone narrow mononuclear cells. See U.S. Pat. Nos. 5,536,475, 5,035,994, 5,130,144, 4,965,205, the contents of each of which is incorporated herein by reference. Two clinical studies support the clinical application of bone marrow derived CD34+ cells after myocardial infarction. See C. Stamm, et al., Lancet 361: 45-46 (2003); Herenstein, B. et al., Blood Supplement, Abs. 2696 (2004).
Animal Models
A preferred therapy after AMI would stop cells from dying during recovery that leads to reverse remodeling and failure, or replace the dying cells with cardiomyocytes.
A conditional transgenic system that allows control of the timing of VEG-F expression has been described in mice. May, D et al, Proc. Nat'l Acad. Sci. 105(1): 282-87 (2008). The system was used to create a tunable state of ventricular hypoperfusion and myocardial ischemia. Under conditions where a large fraction of cardiomyocytes are driven to enter the hibernation mode yet without a detectable cell death, cardiomyocyte dysfunction (hibernation) was found to be linearly related to the extent of reduction in microvascular density. This does not model AMI-induced ventricular remodeling, which is complicated by processes of infarct expansion, inflammation, scar formation and myocyte hypertrophy.
Peripheral artery disease (PAD), also called peripheral vascular disease (PVD), is modeled by the hind limb model of ischemia in which the femoral artery of the mouse is tied off to simulate peripheral artery disease. PAD, which commonly affects the arteries supplying the leg and includes all diseases caused by the obstruction of large arteries in the arms and legs, can result from atherosclerosis, inflammatory processes leading to stenosis, an embolism or thrombus formation. Restriction of blood flow due to arterial stenosis or occlusion often leads patients to complain of muscle pain on walking (intermittent claudication). Any further reduction in blood flow causes ischemic pain at rest. This condition is called chronic limb ischemia, meaning the demand for oxygen cannot be sustained when resting. Ulceration and gangrene may then supervene in the toes, which are the furthest away from the blood supply, and can result in loss of the involved limb if not treated.
Therapies for limb ischemia have the goals of collateral development and blood supply replenishment. Bone marrow derived CD34+ mononuclear cells have been tested in such hindlimb ischemia models, but the hindlimb ischemia model does not model what takes place in the heart.
The closest animal model, the pig model, is not a good model of human disease because (i) all experiments generally are done in nonatherosclerotic animals, (ii) the animals are not treated with angioplasty, (iii) normal pigs do not embolize blood vessels; (iv) circulation of the pig is not exactly the same as human; and (iv) the peri-infarct border zone may not be the same.
A marginal improvement in angina symptoms recently was reported when CD34+ cells were mobilized with GCSF, apheresed after 5 days, and then injected into an ischemic area of the heart based on Noga mapping.
The described invention is a therapy for improving infarct-area perfusion after myocardial infarction. Data from a phase I trial has provided evidence that subjects treated with at least 10×106 isolated autologous CD34+ hematopoietic stem cells containing a subpopulation of at least 0.5×106 potent CD34+ cells expressing CXCR-4 and having CXCR-4 mediated chemotactic activity experienced significant improvement in resting perfusion rates at 6 months compared to subjects receiving 5 million cells and control, as measured by the SPECT Total Severity Score (−256 versus +13, p=0.01).
SUMMARY OF THE INVENTION
The described invention provides pharmaceutical compositions for treating an infarct area injury and methods of treating or repairing the infarct area injury in a revascularized subject in the aftermath of an acute myocardial infarction resulting from a natural disease process by administering to the subject parenterally through a catheter a sterile pharmaceutical composition containing a therapeutically effective amount of a nonexpanded sterile isolated chemotactic hematopoietic stem cell product as a first therapeutic agent and optionally a therapeutically effective amount of at least one compatible second therapeutic agent. The infarct area-improving amount of the sterile isolated chemotactic hematopoietic stem cell product comprises an enriched population of isolated autologous CD34+ cells containing a subpopulation of potent cells expressing CXCR-4 and having CXCR-4-mediated chemotactic activity such that the enriched population of isolated autologous CD34+ hematopoietic stem cells provides at least 0.5×106 potent CD34+ cells expressing CXCR-4 and having CXCR-4 mediated chemotactic activity.
According to one aspect, the described invention provides a method of treating or repairing an infarct area injury in a revascularized subject following an acute myocardial infarction resulting from a natural disease process, the method comprising the steps: (a) administering to the subject parenterally through a catheter a sterile pharmaceutical composition comprising: (i) an infarct area perfusion-improving amount of a nonexpanded sterile isolated chemotactic hematopoietic stem cell product as a first therapeutic agent, wherein the infarct area perfusion-improving amount of the chemotactic hematopoietic stem cell product comprises an enriched population of isolated autologous CD34+ hematopoietic stem cells containing a subpopulation of potent CD34+ cells expressing CXCR-4 and having CXCR-4 mediated chemotactic activity such that the enriched population of isolated autologous CD34+ hematopoietic stem cells provides at least 0.5×106 potent CD34+ cells expressing CXCR-4 and having CXCR-4 mediated chemotactic activity; (ii) a stabilizing amount of serum, wherein the stabilizing amount of serum is greater than 20% (v/v), and (iii) optionally a therapeutically effective amount of at least one compatible second therapeutic agent; and (b) improving perfusion in at least one infarct area, relative to controls, wherein at least 70% of cells in the enriched population of isolated CD34+ cells containing the subpopulation of potent cells that express CXCR-4 and that have CXCR-4-mediated chemotactic activity when passed through the catheter and when tested in vitro are CD34+ cells, and wherein the enriched population of isolated CD34+ cells containing a subpopulation of potent cells that express CXCR-4 and that have CXCR-4-mediated chemotactic activity when passed through the catheter and tested in vitro (1) retains the CXCR-4-mediated chemotactic activity; (2) is at least about 70% viable; and (3) is able to form hematopoietic colonies in vitro, for at least about 24 hours following acquisition from the subject of the enriched population of CD34+ cells containing the subpopulation of potent cells that express CXCR-4; and wherein administering step (a) occurs at one or more infusion dates, and wherein a first infusion date comprises a specific time interval defined by a first time and a second time, wherein the first time is after peak inflammatory cytokine cascade production in the infarcted area and the second time is before myocardial scar formation in the infarcted area. According to one embodiment of the method, the infarct area perfusion-improving amount of the chemotactic hematopoietic stem cell product comprises an enriched population of at least 10×106 isolated autologous CD34+ hematopoietic stem cells containing a subpopulation of 0.5×106 potent CD34+ cells expressing CXCR-4 and having CXCR-4 mediated chemotactic activity. According to another embodiment, the infarct area injury comprises apoptotic cardiomyocyte loss in the infarct area. According to another embodiment, the infarct area injury comprises adverse ventricular remodeling after an acute myocardial infarction, when compared to controls. According to another embodiment, the infarct area injury comprises a progressive decline in heart muscle function following the acute myocardial infarction. According to another embodiment, the infarct area injury comprises hypoperfusion of at least one ischemic peri-infarct zone of myocardial tissue. According to another embodiment, the infarct area injury comprises myocardial hibernation in the peri-infarct border zone. According to another embodiment, the method further comprises the step: administering at a second infusion date a frozen and thawed second aliquot of the second sterile pharmaceutical composition, the frozen and thawed second aliquot comprising (i) a frozen and thawed enriched population of isolated autologous CD34+ hematopoietic stem cells containing a subpopulation of at least 0.5×106 potent CD34+ cells expressing CXCR-4 and having CXCR-4 mediated chemotactic activity; and (ii) a stabilizing amount of serum, wherein the stabilizing amount of serum is greater than 20% (v/v), wherein the frozen and thawed enriched population of isolated CD34+ cells containing the subpopulation of potent cells expressing CXCR-4 and having CXCR-4-mediated chemotactic activity of the second aliquot, when passed through the catheter and tested in vitro, (1) retains the CXCR-4-mediated activity; (2) contains at least 70% CD34+ cells; (3) is at least 70% viable; and (4) is able to form hematopoietic colonies in vitro, for at least about 24 hours following thawing of the second aliquot. According to another embodiment, the method further comprises the step: optionally administering at a third infusion data a frozen and thawed third aliquot of the sterile pharmaceutical composition, the third aliquot comprising a frozen and thawed enriched population of isolated autologous CD34+ hematopoietic stem cells containing a subpopulation of at least 0.5×106 potent CD34+ cells expressing CXCR-4 and having CXCR-4 mediated chemotactic activity; (ii) a stabilizing amount of serum, wherein the stabilizing amount of serum is greater than 20% (v/v), wherein the frozen and thawed enriched population of isolated CD34+ cells containing the subpopulation of potent cells expressing CXCR-4 and having CXCR-4-mediated chemotactic activity of the third aliquot, when passed through the catheter and tested in vitro, (1) retains the CXCR-4-mediated activity; (2) contains at least 70% CD34+ cells; (3) is at least 70% viable; and (4) is able to form hematopoietic colonies in vitro, for at least about 24 hours following thawing of the third aliquot. According to another embodiment, the second infusion date is about 30 days after the first infusion date. According to another embodiment, the third infusion date is about 60 days after the first infusion date. According to another embodiment, the enriched population of CD34+ cells containing a subpopulation of potent cells expressing CXCR-4 and having CXCR-4 mediated chemotactic activity (a) is capable of forming hematopoietic colonies in vitro and (b) retains at least 2% of the CXCR-4-mediated chemotactic activity, for at least 48 hours following acquisition of the of the enriched population of CD34+ cells containing a subpopulation of potent cells that express CXCR-4 in (a). According to another embodiment, the enriched population of CD34+ cells containing a subpopulation of potent cells expressing CXCR-4 and having CXCR-4 mediated chemotactic activity (a) is capable of forming hematopoietic colonies in vitro; and (b) retains at least 2% of the CXCR-4-mediated chemotactic activity, for at least 72 hours following acquisition of the enriched population of CD34+ cells containing a subpopulation of potent cells that express CXCR-4 in (a). According to another embodiment, the subpopulation of potent cells expressing CXCR-4 and having CXCR-4 mediated chemotactic activity retains at least 2% of the CXCR-4-mediated chemotactic activity for at least 24 hours following acquisition from the subject of the enriched population of CD34+ cells containing a subpopulation of potent cells that express CXCR-4 in (a). According to another embodiment, the method further comprises the step of delivering the composition intravascularly to an infarct related artery. According to another embodiment, the method further comprises the step of delivering the composition through the catheter into myocardium. According to another embodiment, the catheter is a flow control catheter. According to another embodiment, the catheter is a balloon dilatation catheter. According to another embodiment, the catheter has an internal diameter of at least about 0.36 mm. According to another embodiment, the first time of the specific time interval of the first infusion date is at least about 5 days post-infarction. According to another embodiment, the second time of the specific time interval of the first infusion date is less than about 14 days post-infarction. According to another embodiment, the first time of the specific time interval of the first infusion date is at least about 5 days post-infarction and the second time of the specific time interval of the first infusion date is less than about 14 days post-infarction. According to another embodiment, the optional second therapeutic agent is at least one compatible agent that promotes cardiomyocyte growth. According to another embodiment, the optional second therapeutic agent is selected from the group consisting of an angiotensin converting enzyme inhibitor, a beta-blocker, a diuretic, an anti-arrhythmic agent, an anti-anginal agent, a tyrosine kinase receptor agonist, a vasoactive agent, an anticoagulant agent, a fibrinolytic agent, and a hypercholesterolemic agent. According to another embodiment, the at least one compatible agent that promotes cardiomyocyte growth comprises the tyrosine kinase receptor agonist neuregulin 1. According to another embodiment, the method reduces the infarct area injury more than components (i) plus (ii) or component (iii) alone. According to another embodiment, the method improves microvascular blood flow in the infarct area, when compared to controls. According to another embodiment, the method decreases area of the infarct injury, when compared to controls. According to another embodiment, the method decreases infarct mass, when compared to controls. According to another embodiment, the method increases perfusion of at least one ischemic peri-infarct zone of myocardial tissue, when compared to controls. According to another embodiment, the method increases perfusion to hibernating myocardium in at least one peri-infarct zone of myocardial tissue, when compared to controls. According to another embodiment, the at least one compatible agent that promotes cardiomyocyte growth comprises a vascular endothelial growth factor selected from the group consisting of VEGF-A, VEGF-B, VEGF-C, and VEGF-D. According to another embodiment, the at least one compatible agent that promotes cardiomyocyte growth comprises placental growth factor. According to another embodiment, the at least one compatible agent that promotes cardiomyoctyte growth comprises a catecholamine. According to another embodiment, the catecholamine is norepinephrine. According to another embodiment, the at least one compatible agent that promotes cardiomyocyte growth comprises endothelin-1. According to another embodiment, the at least one compatible agent that promotes cardiomyocyte growth comprises a prostaglandin F2α. According to another embodiment, the at least one compatible agent that promotes cardiomyocyte growth is angiotensin II. According to another embodiment, the at least one compatible agent that promotes cardiomyocyte growth comprises a phorbol ester. According to another embodiment, the at least one compatible agent that promotes cardiomyocyte growth comprises neuropeptide Y. According to another embodiment, the at least one compatible agent that promotes cardiomyocyte growth comprises active transforming growth factor β1. the at least one compatible agent that promotes cardiomyocyte growth comprises Gq protein. According to another embodiment, the at least one compatible agent that promotes cardiomyocyte growth comprises diacyl glyercol. According to another embodiment, the at least one compatible agent that promotes cardiomyocyte growth comprises salusin-α. According to another embodiment, the at least one compatible agent that promotes cardiomyocyte growth comprises salusin-β. According to another embodiment, the at least one compatible agent that promotes cardiomyocyte growth comprises insulin-like growth factor-1. According to another embodiment, the at least one compatible agent that promotes cardiomyocyte growth comprises myostatin. According to another embodiment, the at least one compatible agent that promotes cardiomyocyte growth comprises granulocyte colony-stimulating factor. According to another embodiment, the at least one compatible agent that promotes cardiomyocyte growth comprises macrophage colony-stimulating factor. According to another embodiment, the at least one compatible agent that promotes cardiomyocyte growth comprises tumor necrosis factor-like weak inducer of apoptosis (TWEAK). According to another embodiment, the at least one compatible agent that promotes cardiomyocyte growth comprises a thiazolidinedione. According to another embodiment, the thiazolidinedione is rosiglitazone.
According to another aspect, the described invention provides a pharmaceutical composition for treating an infarct area injury of a revascularized subject following an acute myocardial infarction resulting from a natural disease process, comprising: (a) an infarct-injury improving amount of a sterile isolated chemotactic hematopoietic stem cell product, wherein the infarct area-improving amount of the sterile isolated chemotactic hematopoietic stem cell product comprises an enriched population of isolated autologous CD34+ cells containing a subpopulation of potent cells expressing CXCR-4 and having CXCR-4-mediated chemotactic activity such that the enriched population of isolated autologous CD34+ hematopoietic stem cells provides at least 0.5×106 potent CD34+ cells expressing CXCR-4 and having CXCR-4 mediated chemotactic activity; (b) a stabilizing amount of serum, wherein the stabilizing amount of serum is greater than 20% (v/v), and (c) a therapeutically effective amount of at least one compatible agent that promotes cardiomyocyte growth, wherein the composition is administered to the subject parenterally through a catheter; wherein at least 70% of cells in the enriched population of isolated CD34+ cells containing the subpopulation of potent cells that express CXCR-4 and that have CXCR-4-mediated chemotactic activity when passed through the catheter and when tested in vitro are CD34+ cells, and wherein the enriched population of isolated CD34+ cells containing a subpopulation of potent cells that express CXCR-4 and that have CXCR-4-mediated chemotactic activity when passed through the catheter and tested in vitro: (1) retains the CXCR-4-mediated chemotactic activity; (2) is at least about 70% viable; and (3) is able to form hematopoietic colonies in vitro, for at least about 24 hours following acquisition from the subject of the enriched population of CD34+ cells containing the subpopulation of potent cells that express CXCR-4. According to one embodiment, the infarct area perfusion-improving amount of the chemotactic hematopoietic stem cell product comprises an enriched population of at least 10×106 isolated autologous CD34+ hematopoietic stem cells containing a subpopulation of 0.5×106 potent CD34+ cells expressing CXCR-4 and having CXCR-4 mediated chemotactic activity. According to another embodiment, the composition reduces the infarct area injury more than components (i) plus (ii) or component (iii) alone. According to another embodiment, the infarct area injury comprises apoptotic cardiomyocyte loss in the infarct area. According to another embodiment, the infarct area injury comprises adverse ventricular remodeling after an acute myocardial infarction, when compared to controls. According to another embodiment, the infarct area injury comprises a progressive decline in heart muscle function following the acute myocardial infarction. According to another embodiment, the infarct area injury comprises hypoperfusion of at least one ischemic peri-infarct zone of myocardial tissue. According to another embodiment, the infarct area injury comprises myocardial hibernation in the peri-infarct border zone. According to another embodiment, the enriched population of isolated CD34+ cells containing a subpopulation of potent cells expressing CXCR-4 and having CXCR-4-mediated chemotactic activity is purified from cellular components of a bone marrow aspirate acquired from the subject. According to another embodiment, the enriched population of isolated CD34+ cells containing a subpopulation of potent cells expressing CXCR-4 and having CXCR-4-mediated chemotactic activity is purified from peripheral blood. According to another embodiment, the enriched population of CD34+ cells containing a subpopulation of potent cells that express CXCR-4 and that have CXCR-4-mediated chemotactic activity (a) is capable of forming hematopoietic colonies in vitro; and (b) retains at least 2% of the CXCR-4-mediated chemotactic activity, for at least about 48 hours following acquisition from the subject of the enriched population of CD34+ cells containing the subpopulation of potent cells that express CXCR-4 in (a). According to another embodiment, the enriched population of CD34+ cells containing a subpopulation of potent cells that express CXCR-4 and that have CXCR-4-mediated chemotactic activity (a) is capable of forming hematopoietic colonies in vitro; and (b) retains at least 2% of the CXCR-4-mediated chemotactic activity, for at least about 72 hours following acquisition from the subject of the enriched population of CD34+ cells containing the subpopulation of potent cells that express CXCR-4 in (a). According to another embodiment, the enriched population of CD34+ cells containing a subpopulation of potent cells that express CXCR-4 and that have CXCR-4-mediated chemotactic activity retains at least 2% of the CXCR-4-mediated chemotactic activity for at least about 24 hours following acquisition from the subject of the enriched population of CD34+ cells containing the subpopulation of potent cells that express CXCR-4 in (a). According to another embodiment, the composition is administered through the catheter intravascularly to an infarct-related artery. According to another embodiment, the composition is administered through the catheter into myocardium. According to another embodiment, the composition improves microvascular blood flow in the infarct area, when compared to controls. According to another embodiment, the composition increases perfusion of at least one ischemic peri-infarct zone of myocardial tissue, when compared to controls. According to another embodiment, the composition increases perfusion to hibernating myocardium in at least one peri-infarct zone of myocardial tissue, when compared to controls. According to another embodiment, the composition decreases area of infarct area injury, when compared to controls. According to another embodiment, the composition decreases infarct mass, when compared to controls. According to another embodiment, the at least one compatible agent that promotes cardiomyocyte growth is selected from the group consisting of an angiotensin converting enzyme inhibitor, a beta-blocker, a diuretic, an anti-arrhythmic agent, an anti-anginal agent, a tyrosine kinase receptor agonist, a vasoactive agent, an anticoagulant agent, a fibrinolytic agent, and a hypercholesterolemic agent. According to another embodiment, the at least one compatible agent that promotes cardiomyocyte growth comprises the tyrosine kinase receptor agonist neuregulin 1. According to another embodiment, the at least one compatible agent that promotes cardiomyocyte growth comprises an vascular endothelial growth factor selected from the group consisting of VEGF-A. VEGF-B, VEGF-C, and VEGF-D. According to another embodiment, the at least one compatible agent that promotes cardiomyocyte growth comprises placental growth factor. According to another embodiment, the at least one compatible agent that promotes cardiomyoctyte growth comprises a catecholamine. According to another embodiment, the catecholamine is norepinephrine. According to another embodiment, the at least one compatible agent that promotes cardiomyocyte growth comprises endothelin-1. According to another embodiment, the at least one compatible agent that promotes cardiomyocyte growth comprises a prostaglandin F2α. According to another embodiment, the at least one compatible agent that promotes cardiomyocyte growth comprises angiotensin II According to another embodiment, the at least one compatible agent that promotes cardiomyocyte growth comprises a phorbol ester. According to another embodiment, the at least one compatible agent that promotes cardiomyocyte growth comprises neuropeptide Y. According to another embodiment, the at least one compatible agent that promotes cardiomyocyte growth comprises active transforming growth factor β1. According to another embodiment, the at least one compatible agent that promotes cardiomyocyte growth comprises Gq protein. According to another embodiment, the at least one compatible agent that promotes cardiomyocyte growth comprises diacyl glyercol (DAG). According to another embodiment, the at least one compatible agent that promotes cardiomyocyte growth comprises salusin-α. According to another embodiment, the at least one compatible agent that promotes cardiomyocyte growth comprises salusin-β. According to another embodiment, the at least one compatible agent that promotes cardiomyocyte growth comprises insulin-like growth factor-1. According to another embodiment, the at least one compatible agent that promotes cardiomyocyte growth comprises myostatin. According to another embodiment, the at least one compatible agent that promotes cardiomyocyte growth comprises granulocyte colony-stimulating factor. According to another embodiment, the at least one compatible agent that promotes cardiomyocyte growth comprises macrophage colony-stimulating factor. According to another embodiment, the at least one compatible agent that promotes cardiomyocyte growth comprises tumor necrosis factor-like weak inducer of apoptosis (TWEAK). According to another embodiment, the at least one compatible agent that promotes cardiomyocyte growth comprises a thiazolidinedione. According to another embodiment, the thiazolidinedione is rosiglitazone.



BRIEF DESCRIPTION OF THE FIGURES
FIG. 1 shows that the functional viability of the chemotactic hematopoietic cell product of the invention at 72 hours is equivalent to that at 48 hours.
FIG. 2 shows the migratory efficiency of the formulated chemotactic hematopoietic stem cell product comprising CD34+ cells of the invention.
FIG. 3 shows the improved stability of CD34+ cells formulated in human serum.
FIG. 4(A) shows the change in infarct size as a percent of LV mass versus the product (CD34+ dose times % of CD34+ cells mobile in an SDF gradient). FIG. 4(B) shows the change in perfusion defect (RTSS) versus the product (CD34+ dose time % of CD34+ cells mobile in an SDF gradient).



DETAILED DESCRIPTION
The invention provides infarct area perfusion-improving compositions comprising a nonexpanded sterile isolated chemotactic hematopoietic stem cell product and methods of use thereof to treat an infarction area injury and improve cardiac function in the aftermath of an acute myocardial infarction resulting from a natural disease process in a revascularized subject.
Glossary
The term “transforming growth factor β1” (TGF-β1) refers to a multifunctional protein that controls proliferation, differentiation and other functions in many cell types. Many cells synthesize TGF-β1 and have specific receptors for it. It positively and negatively regulates many other growth factors, and as a potent stimulator of osteoblastic bone formation, causing chemotaxis, proliferation and differentiation in committed osteoblasts, has an important role in bone remodeling (Schluter, K. D., and Piper, H. M. FASEB J. 13:S17-S-22 (1999)).
The term “administer” as used herein in its various grammatical forms means to give or to apply. The term “administering” as used herein includes in vivo administration, as well as administration directly to tissue ex vivo. Generally, compositions may be administered systemically either parenterally or topically in dosage unit formulations containing conventional nontoxic pharmaceutically acceptable carriers, adjuvants, and vehicles as desired, or may be locally administered by means such as, but not limited to, injection, implantation, grafting, topical application, or parenterally. A means of administering cells may include, but is not limited to, infusion.
The term “aftermath” as used herein refers to a consequence or outcome that results or follows from an event.
As used herein, the term “angiogenesis” refers to the process of formation and development of blood vessels.
The term “angiotensin II” refers to a polypeptide hormone that is formed from angiotensin I by the action of angiotensin-converting enzyme (or ACE).
The terms “apoptosis” or “programmed cell death” refer to a highly regulated and active process that contributes to biologic homeostasis comprised of a series of biochemical events that lead to a variety of morphological changes, including blebbing, changes to the cell membrane, such as loss of membrane asymmetry and attachment, cell shrinkage, nuclear fragmentation, chromatin condensation, and chromosomal DNA fragmentation, without damaging the organism.
Apoptotic cell death is induced by many different factors and involves numerous signaling pathways, some dependent on caspase proteases (a class of cysteine proteases) and others that are caspase independent. It can be triggered by many different cellular stimuli, including cell surface receptors, mitochondrial response to stress, and cytotoxic T cells, resulting in activation of apoptotic signaling pathways
The caspases involved in apoptosis convey the apoptotic signal in a proteolytic cascade, with caspases cleaving and activating other caspases that then degrade other cellular targets that lead to cell death. The caspases at the upper end of the cascade include caspase-8 and caspase-9. Caspase-8 is the initial caspase involved in response to receptors with a death domain (DD) like Fas.
Receptors in the TNF receptor family are associated with the induction of apoptosis, as well as inflammatory signaling. The Fas receptor (CD95) mediates apoptotic signaling by Fas-ligand expressed on the surface of other cells. The Fas-FasL interaction plays an important role in the immune system and lack of this system leads to autoimmunity, indicating that Fas-mediated apoptosis removes self-reactive lymphocytes. Fas signaling also is involved in immune surveillance to remove transformed cells and virus infected cells. Binding of Fas to oligomerized FasL on another cell activates apoptotic signaling through a cytoplasmic domain termed the death domain (DD) that interacts with signaling adaptors including FAF, FADD and DAX to activate the caspase proteolytic cascade. Caspase-8 and caspase-10 first are activated to then cleave and activate downstream caspases and a variety of cellular substrates that lead to cell death.
Mitochondria participate in apoptotic signaling pathways through the release of mitochondrial proteins into the cytoplasm. Cytochrome c, a key protein in electron transport, is released from mitochondria in response to apoptotic signals, and activates Apaf-1, a protease released from mitochondria. Activated Apaf-1 activates caspase-9 and the rest of the caspase pathway. Smac/DIABLO is released from mitochondria and inhibits IAP proteins that normally interact with caspase-9 to inhibit apoptosis. Apoptosis regulation by Bcl-2 family proteins occurs as family members form complexes that enter the mitochondrial membrane, regulating the release of cytochrome c and other proteins. TNF family receptors that cause apoptosis directly activate the caspase cascade, but can also activate Bid, a Bcl-2 family member, which activates mitochondria-mediated apoptosis. Bax, another Bcl-2 family member, is activated by this pathway to localize to the mitochondrial membrane and increase its permeability, releasing cytochrome c and other mitochondrial proteins. Bcl-2 and Bcl-xL prevent pore formation, blocking apoptosis. Like cytochrome c, AIF (apoptosis-inducing factor) is a protein found in mitochondria that is released from mitochondria by apoptotic stimuli. While cytochrome C is linked to caspase-dependent apoptotic signaling, AIF release stimulates caspase-independent apoptosis, moving into the nucleus where it binds DNA. DNA binding by AIF stimulates chromatin condensation, and DNA fragmentation, perhaps through recruitment of nucleases.
The mitochondrial stress pathway begins with the release of cytochrome c from mitochondria, which then interacts with Apaf-1, causing self-cleavage and activation of caspase-9. Caspase-3, -6 and -7 are downstream caspases that are activated by the upstream proteases and act themselves to cleave cellular targets.
Granzyme B and perforin proteins released by cytotoxic T cells induce apoptosis in target cells, forming transmembrane pores, and triggering apoptosis, perhaps through cleavage of caspases, although caspase-independent mechanisms of Granzyme B mediated apoptosis have been suggested.
Fragmentation of the nuclear genome by multiple nucleases activated by apoptotic signaling pathways to create a nucleosomal ladder is a cellular response characteristic of apoptosis. One nuclease involved in apoptosis is DNA fragmentation factor (DFF), a caspase-activated DNAse (CAD). DFF/CAD is activated through cleavage of its associated inhibitor ICAD by caspases proteases during apoptosis. DFF/CAD interacts with chromatin components such as topoisomerase II and histone H1 to condense chromatin structure and perhaps recruit CAD to chromatin. Another apoptosis activated protease is endonuclease G (EndoG). EndoG is encoded in the nuclear genome but is localized to mitochondria in normal cells. EndoG may play a role in the replication of the mitochondrial genome, as well as in apoptosis. Apoptotic signaling causes the release of EndoG from mitochondria. The EndoG and DFF/CAD pathways are independent since the EndoG pathway still occurs in cells lacking DFF.
Hypoxia, as well as hypoxia followed by reoxygenation can trigger cytochrome c release and apoptosis. Glycogen synthase kinase (GSK-3) a serine-threonine kinase ubiquitously expressed in most cell types, appears to mediate or potentiate apoptosis due to many stimuli that activate the mitochondrial cell death pathway. Loberg, R D, et al., J. Biol. Chem. 277 (44): 41667-673 (2002). It has been demonstrated to induce caspase 3 activation and to activate the proapoptotic tumor suppressor gene p53. It also has been suggested that GSK-3 promotes activation and translocation of the proapoptotic Bcl-2 family member, Bax, which, upon aggregation and mitochondrial localization, induces cytochrome c release. Akt is a critical regulator of GSK-3, and phosphorylation and inactivation of GSK-3 may mediate some of the antiapoptotic effects of Akt.
The term “biomarker” as used herein refers to an objectively measured indicator of disease risk, underlying pathobiological processes, diagnosis and stage of disease, prognosis, treatment response, recurrence, and clinical outcomes. A biomarker can take the form of a gene, genetic variations, RNA proteins and metabolites. Biomarkers that reliably mirror or predict the progression or amelioration of a disease may assist in disease diagnosis and assessment of disease severity, risk of onset, and progression.
The term “c-kit” refers to a protein on the surface of some cells that binds to stem cell factor (a substance that causes certain types of cells to grow). Altered forms of this receptor may be associated with some types of cancer.
The term “cardiac biomarkers” refers to enzymes, proteins and hormones associated with heart function, damage or failure that are used for diagnostic and prognostic purposes. Different cardiac biomarkers have different times that their levels rise, peak, and fall within the body, allowing them to be used, not only to track the progress of a heart attack, but to estimate when it began and to monitor for recurrence. Some of the tests are specific for the heart while others also are elevated with skeletal muscle damage. Current cardiac biomarkers include, but are not limited to CK (creatine phosphokinase or creatine kinase) and CK-MB (creatine kinase-myoglobin levels (to help distinguish between skeletal and heart muscle)), troponin (blood levels of troponin I or T will remain high for 1-2 weeks after a heart attack; troponin generally is not affected by damage to other muscles), myoglobin (to determine whether muscle, particularly heart muscle, has been injured), and BNP (brain natriuretic peptide) or NT-proBNP (N-terminal prohormone brain natriuretic peptide (to help diagnose heart failure and grade the severity of that heart failure).
The term “cardiac catheterization” refers to a procedure in which a catheter is passed through an artery to the heart, and into a coronary artery. This procedure produces angiograms (i.e., x-ray images) of the coronary arteries and the left ventricle, the heart's main pumping chamber, which can be used to measure pressures in the pulmonary artery, and to monitor heart function.
The term “catecholamine” refers to any of a class of amines that possess a catechol (C6H4(OH2)) ring. Nonlimiting examples of catecholamines include dopamine, adrenaline, and norepinephrine (noradrenaline).
The cluster of differentiation (cluster of designation) (often abbreviated as CD) is a protocol used for the identification and investigation of cell surface molecules present on white blood cells. CD molecules can act in numerous ways, often acting as receptors or ligands (meaning the molecule that activates a receptor) important to the cell. A signal cascade usually is initiated, altering the behavior of the cell. Some CD proteins do not play a role in cell signaling, but have other functions, such as cell adhesion.
The term “cell surface marker” as used herein refers to an antigenic determinant or epitope found on the surface of a specific type of cell. Cell surface markers can facilitate the characterization of a cell type, its identification, and eventually its isolation.
The term “CD34+ cells” as used herein refers to hematopoietic stem and progenitor cells derived from human bone marrow that “are positive for” i.e., “express”, a hematopoietic stem cell antigen, at least a subpopulation of which express CXCR4, and that can migrate to areas of injury.
The term “CD38” refers to a protein marker present on macrophages, dendritic cells, and activated B and NK cells, which may mediate the adhesion between lymphocytes and endothelial cells.
The terms “CD45” and “common leukocyte antigen” refer to a protein tyrosine phosphatase (PTP) located in hematopoietic cells except erythrocytes and platelets.
The term “CD59” refers to a glycosylphosphatidylinositol (GPI)-linked membrane glycoprotein, which protects human cells from complement-mediated lysis.
The term “CXCR-4” as used herein refers to a G-protein-linked chemokine receptor.
The term “cytokine” as used herein refers to small soluble protein substances secreted by cells, which have a variety of effects on other cells. Cytokines mediate many important physiological functions including growth, development, wound healing, and the immune response. They act by binding to their cell-specific receptors located in the cell membrane, which allows a distinct signal transduction cascade to start in the cell, which eventually will lead to biochemical and phenotypic changes in target cells. Generally, cytokines act locally. They include type I cytokines, encompassing many of the interleukins, as well as several hematopoietic growth factors; type II cytokines, including the interferons and interleukin-10; tumor necrosis factor (“TNF”)-related molecules, including TNFα and lymphotoxin; immunoglobulin super-family members, including interleukin 1 (“IL-1”); and the chemokines, a family of molecules that play a critical role in a wide variety of immune and inflammatory functions. The same cytokine can have different effects on a cell depending on the state of the cell. Cytokines often regulate the expression of, and trigger cascades of, other cytokines.
The term “colony stimulating factor” refers to a cytokine responsible for controlling the production of white blood cells. Types include granulocyte colony stimulating factor (G-CSF), macrophage colony stimulating factor (M-CSF), and granulocyte macrophage colony stimulating factor (GM-CSF).
The term “diacyl glycerol” (DAG) refers to a glyceride consisting of two fatty acid chains covalently bonded to a glycerol molecule through ester linkages. Diacylglycerols also can have many different combinations of fatty acids attached at both the C1 and C2 positions.
The term “endothelin” refers to a vasoconstrictive peptide that is synthesized and released by vascular endothelium and that is a marker of endothelial function.
The terms “formulation” and “composition” are used interchangeably herein to refer to a product of the described invention that comprises all active and inert ingredients. The term “active” refers to the ingredient, component or constituent of the compositions of the described invention responsible for the intended therapeutic effect. The terms “pharmaceutical formulation” or “pharmaceutical composition” as used herein refer to a formulation or composition that is employed to prevent, reduce in intensity, cure or otherwise treat a target condition or disease.
The term “Gq” protein refers to a heterotrimeric G protein subunit that activates phopholipase C and participates in a variety of cellular signaling pathways.
The term “hematopoietic stem cell” refers to a cell isolated from blood or from bone marrow that can renew itself, differentiate to a variety of specialized cells, mobilize out of the bone marrow into the circulating blood, and undergo programmed cell death (apoptosis). In some embodiments of the described invention, hematopoietic stem cells derived from human subjects express at least one type of cell surface marker, including, but not limited to, CD34, CD38, HLA-DR, c-kit, CD59, Sca-1, Thy-1, and/or CXCR-4, or a combination thereof.
“HLA-DR” refers to a human class II histocompatibility antigen present on several cell types, including antigen-presenting cells, B cells, monocytes, macrophages, and activated T cells.
The term “insulin-like growth factor 1” (IGF-1) refers to a protein similar to insulin in function and structure that is a member of a family of proteins involved in mediating growth and development.
The term “isolate” and its various grammatical forms as used herein refers to placing, setting apart, or obtaining a protein, molecule, substance, nucleic acid, peptide, cell or particle, in a form essentially free from contaminants or other materials with which it is commonly associated.
The term “interleukin” as used herein refers to a cytokine secreted by white blood cells as a means of communication with other white blood cells.
The terms “VEGF”, “VEGF-1” or “vascular endothelial growth factor-1” are used interchangeably herein to refer to a cytokine that mediates numerous functions of endothelial cells including proliferation, migration, invasion, survival, and permeability. The term “VEGF-2” refers to a regulator for growth of vascular endothelial and smooth muscle cells. VEGF-2 stimulates the growth of human vascular endothelial cells but inhibits growth of human aortic smooth muscle cells induced by platelet-derived growth factor.
The term “chemokine” as used herein refers to a class of chemotactic cytokines that signal leukocytes to move in a specific direction.
The terms “chemotaxis” or “chemotactic” refer to the directed motion of a motile cell or part along a chemical concentration gradient towards environmental conditions it deems attractive and/or away from surroundings it finds repellent. In one aspect of the described invention, the potent CD34+CXCR-4+ cells of the described invention can migrate, meaning that they can move from one place, location or area to another. In one embodiment, their migration is driven by chemotaxis.
The term “complete blood count” (CBC) refers to a laboratory test that provides detailed information about the amount and the quality of each of the blood cell types. It usually includes a measurement of each of the three major blood cells (red blood cells, white blood cells, and platelets) and a measure of the hemoglobin and hematocrit. “Hemoglobin” (HGB) refers to the number of grams of hemoglobin in a deciliter of blood (g/dL). Normal hemoglobin levels in healthy adult human subjects are about 14 g/dL to about 18 g/dL for men and about 12 g/dL to about 16 g/dL for women. As a rough guideline, hemoglobin generally should be about one-third the hematocrit. “Hematocrit” (HCT) refers to the proportion of red blood cells as a percentage of total blood volume. A normal hematocrit for human subjects is about 40% to about 55% for men and about 35% to about 45% for women. “Red Blood Cell Count” (RBC) refers to the total number of red blood cells in a quantity of blood. Normal ranges in human subjects are about 4.5 million cells/mm3 to about 6.0 million cells/mm3 for men and about 4.0 million cells/mm3 to about 5.5 million cells/mm3 for women. “White Blood Cell Count” (WBC) refers to the total number of while blood cells or leukocytes in a quantity of blood. Normal ranges in human subjects are about 4.3×103 cells/mm3 to about 10.8×103 cells/mm3.
The term “disease” or “disorder”, as used herein, refers to an impairment of health or a condition of abnormal functioning. The term “syndrome,” as used herein, refers to a pattern of symptoms indicative of some disease or condition. The term “condition”, as used herein, refers to a variety of health states and is meant to include disorders or diseases caused by any underlying mechanism or disorder, injury, and the promotion of healthy tissues and organs.
As used herein, the term “inflammation” refers to a response to infection and injury in which cells involved in detoxification and repair are mobilized to the compromised site by inflammatory mediators.
Regardless of the initiating agent, the physiologic changes accompanying acute inflammation encompass four main features: (1) vasodilation, which results in a net increase in blood flow, is one of the earliest physical responses to acute tissue injury; (2) in response to inflammatory stimuli, endothelial cells lining the venules contract, widening the intracellular junctions to produce gaps, leading to increased vascular permeability, which permits leakage of plasma proteins and blood cells out of blood vessels; (3) inflammation often is characterized by a strong infiltration of leukocytes at the site of inflammation, particularly neutrophils (polymorphonuclear cells). These cells promote tissue damage by releasing toxic substances at the vascular wall or in uninjured tissue; and (4) fever, produced by pyrogens released from leukocytes in response to specific stimuli.
During the inflammatory process, soluble inflammatory mediators of the inflammatory response work together with cellular components in a systemic fashion in the attempt to contain and eliminate the agents causing physical distress. The terms “inflammatory” or immuno-inflammatory” as used herein with respect to mediators refers to the molecular mediators of the inflammatory process. These soluble, diffusible molecules act both locally at the site of tissue damage and infection and at more distant sites. Some inflammatory mediators are activated by the inflammatory process, while others are synthesized and/or released from cellular sources in response to acute inflammation or by other soluble inflammatory mediators. Examples of inflammatory mediators of the inflammatory response include, but are not limited to, plasma proteases, complement, kinins, clotting and fibrinolytic proteins, lipid mediators, prostaglandins, leukotrienes, platelet-activating factor (PAF), peptides and amines, including, but not limited to, histamine, serotonin, and neuropeptides, and proinflammatory cytokines, including, but not limited to, interleukin-1, interleukin-4, interleukin-6, interleukin-S, tumor necrosis factor (TNF), interferon-gamma, and interleukin 12.
The term “in-date” refers to the time interval between completion of acquiring from the subject a preparation comprising an enriched population of potent CD34+ cells under sterile conditions and initiating sterilely purifying potent CD34+ cells from the preparation. The term “out-date” refers to the time interval between completion of acquiring from the subject a preparation comprising an enriched population of potent CD34+ cells under sterile conditions and infusing the formulated pharmaceutical composition comprising a chemotactic hematopoietic cell product into the subject.
The terms “infuse” or “infusion” as used herein refer to the introduction of a fluid other than blood into a blood vessel of a subject, including humans, for therapeutic purposes.
The “infusion solution” of the described invention without serum contains phosphate buffered saline (PBS) supplemented with 25 USP units/ml of heparin and 1% human serum albumin (HSA). In some embodiments, the infusion solution is supplemented with serum. In some embodiments, the serum is autologous.
The term “injury” refers to damage or harm caused to the structure or function of the body of a subject caused by an agent or force, which may be physical or chemical. The term “vascular injury” refers to injury to the vasculature (i.e., the vascular network, meaning the network of blood vessels or ducts that convey fluids, such as, without limitation, blood or lymph).
The term “limit” as used herein refers to restricting or confining an extent, degree, or amount.
The term “macrophage” as used herein refers to a mononuclear, actively phagocytic cell arising from monocytic stem cells in the bone marrow. These cells are widely distributed in the body and vary in morphology and motility. Phagocytic activity typically is mediated by serum recognition factors, including certain immunoglobulins and components of the complement system, but also may be nonspecific. Macrophages also are involved in both the production of antibodies and in cell-mediated immune responses, particularly in presenting antigens to lymphocytes. They secrete a variety of immunoregulatory molecules.
The terms “microbe” or “microorganism” are used interchangeably herein to refer to an organism too small to be seen clearly with the naked eye, including, but not limited to, microscopic bacteria, fungi (molds), algae, protozoa, and viruses.
The term “modulate” in its various grammatical forms as used herein means to regulate, alter, adapt, manipulate, or adjust a certain measure or proportion. Such modulation may be any change, including an undetectable change.
The term “myocardial infarction” refers to death or permanent damage to heart muscle. Most heart attacks are caused by blockage of coronary arteries that interrupts flow of blood and oxygen to the heart muscle, leading to death of heart cells in that area. The damaged heart muscle loses its ability to contract, leaving the remaining heart muscle to compensate for the weakened area. The described invention includes steps related to evaluating the suitability of subjects for treatment according to the described invention by using tests to look at the size, shape, and function of the heart as it is beating, to detect changes to the rhythm of the heart, and to detect and evaluate damaged tissues and blocked arteries. Examples of such tests include, but are not limited to, electrocardiography, echocardiography, coronary angiography, and nuclear ventriculography. Cardiac biomarkers also are used to evaluate the suitability of subjects for treatment according to the described invention.
The term “myostatin” (MSTN) refers to a protein that is a member of the bone morphogenetic protein (BMP) family and the TGF-β superfamily. This group of proteins is characterized by a polybasic proteolytic processing site, which is cleaved to produce a mature protein containing seven conserved cysteine residues. The members of this family are regulators of cell growth and differentiation in both embryonic and adult tissues.
The term “neuropeptide Y” refers to a neuropeptide widely expressed in the central nervous system and that influences many physiological processes, including cortical excitability, stress response, food intake, circadian rhythms, and cardiovascular function. It functions through G protein-coupled receptors to inhibit adenylyl cyclase, to inhibit activated mitogen-activated protein kinase (MAPK), to regulate intracellular calcium levels, and to activate potassium channels.
The term “perfusion” as used herein refers to the process of nutritive delivery of arterial blood to a capillary bed in biological tissue. Perfusion (“F”) can be calculated with the formula F=((PA−PV)/R) wherein PA is mean arterial pressure, PV is mean venous pressure, and R is vascular resistance. Tissue perfusion can be measured in vivo, by, for example, but not limited to, magnetic resonance imaging (MRI) techniques. Such techniques include using an injected contrast agent and arterial spin labeling (ASL) (wherein arterial blood is magnetically tagged before it enters into the tissue of interest and the amount of labeling is measured and compared to a control recording).
The term “placental growth factor” (PIGF) refers to a cytokine that is a member of the vascular endothelial growth factor family.
The term “phorbol ester” refers to a natural, plant-derived organic compound that is a member of the tigliane family of diterpenes.
As used herein, the term “potent” or “potency” refers to the necessary biological activity of the chemotactic hematopoietic stem cell product of the described invention, i.e., potent CD34+CXCR-4+ cells of the described invention remain viable, are capable of CXCR-4-mediated chemotactice mobility, and are able to grow, i.e., to form hematopoietic colonies in an in vitro CFU assay.
The term “prostaglandin” as used herein refers to any of a group of physiologically active substances that are derivatives of prostanoic acid, a 20 carbon structure, which is a fatty acid containing a 5-membered cyclopentane ring.
The term “progenitor cell” as used herein refers to an immature cell in the bone marrow that may be isolated by growing suspensions of marrow cells in culture dishes with added growth factors. Progenitor cells mature into precursor cells that mature into blood cells. Progenitor cells are referred to as colony-forming units (CFU) or colony-forming cells (CFC). The specific lineage of a progenitor cell is indicated by a suffix, such as, but not limited to, CFU-E (erythrocytic), CFU-GM (granulocytic/macrophage), and CFU-GEMM (pluripotent hematopoietic progenitor).
The term “reduce” or “reducing” when used it its various grammatical forms is used herein to refer to lessening, narrowing or bringing down to a smaller extent, size, amount, degree, or intensity.
The term “repair” as used herein as a noun refers to any correction, reinforcement, reconditioning, remedy, making up for, making sound, renewal, mending, patching, or the like that restores function. When used as a verb, it means to correct, to reinforce, to recondition, to remedy, to make up for, to make sound, to renew, to mend, to patch or to otherwise restore function. In some embodiments “repair” includes full repair and partial repair.
The term “salusin” refers to a bioactive peptide of 28 amino acids (salusin-α) or 20 amino acids (salusin-β) that is translated from an alternatively spliced mRNA of TOR2A, a gene encoding a protein of the torsion dystonia family. Salusins increase intracellular Ca2+, upregulate a variety of genes, and induce cell mitogenesis.
The term “Sca-1” or “stem cell antigen-1” refers to a surface protein component in a signaling pathway that affects the self-renewal ability of mesenchymal stem cells.
The term “stem cells” refers to undifferentiated cells having high proliferative potential with the ability to self-renew that can generate daughter cells that can undergo terminal differentiation into more than one distinct cell phenotype.
The term “stent” is used to refer to a small tube used to prop open an artery. The stent is collapsed to a small diameter, put over a balloon catheter, inserted through a main artery in the groin (femoral artery) or arm (brachial artery) and threaded up to the narrowed/blocked section of the artery. When it reaches the right location, the balloon is inflated slightly to push any plaque out of the way and to expand the artery (balloon angioplasty). When the balloon is inflated, the stent expands, locks in place, and forms a scaffold to hold the artery open. The stent stays in the artery permanently. In certain subjects, a stent reduces the renarrowing that occurs after balloon angioplasty or other procedures that use catheters. A stent also may help restore normal blood flow and keep an artery open if it has been torn or injured by the balloon catheter. Reclosure (restenosis) may be a problem with the stent procedure. Drug-eluting stents are stents coated with drugs that are slowly released. These drugs may help keep the blood vessel from reclosing.
The terms “subject” and “patients” are used interchangeably herein and include animal species of mammalian origin, including humans.
The term a “susceptible subject” as used herein refers to a member of a population at risk.
The term “thiazolidinediones” (TZDs) refers to a class of compounds that bind to peroxisome proliferator-activated receptors (PPARs), a group of receptor molecules inside the cell nucleus. The members of this class of compounds are derivatives of the parent compound thiazolidinedione and include rosiglitazone, pioglitazone, and troglitazone.
The term “Thy-1” refers to the Ig superfamily cell surface glycoprotein Thy-1 expressed on immune cells and neurons of rodents and humans, which is hypothesized to function in cell adhesion and signal transduction in T cell differentiation, proliferation, and apoptosis.
As used herein the terms “treat” or “treating” are used interchangeably to include abrogating (abolish, do away with), substantially inhibiting, slowing or reversing the progression of a condition, substantially ameliorating (improving) clinical or aesthetical symptoms of a condition, substantially preventing the appearance of clinical or aesthetical symptoms of a condition, protecting from harmful stimuli, as well as to accomplishing one or more of the following: (a) reducing the severity of a disorder disease or condition; (b) limiting development of symptoms characteristic of the disorder, disease or condition being treated; (c) limiting worsening of symptoms characteristic of the disorder, disease or condition being treated; (d) limiting recurrence of the disorder, disease or condition in patients that have previously had the disorder, disease or condition; and (e) limiting recurrence of symptoms in patients that were previously asymptomatic for the disorder, disease or condition.
The term “tumor necrosis factor-like weak inducer of apoptosis” (TWEAK) refers to a member of the TNF-α growth factor family that is produced as a type II transmembrane protein and is processed into the 156-amino-acid soluble cytokine. (Chicheportiche, Y. et al., J. Biol. Chem. 272: 32401-410 (1997)). TWEAK has multiple biological activities, including stimulation of cell growth and angiogenesis, induction of inflammatory cytokines, and under some experimental conditions, stimulation of apoptosis, (Wiley, S. R., et al. Cytokine Growth Factor Rev. 14(3-4):241-9, 2003) and is a positive regulator of cardiomyocyte proliferation (Novoyatieva, T., et al. Cardiovacs. Res. 2009 Nov. 26, PMID: 19887380). TWEAK mediates these processes through the fibroblast growth factor-inducible molecule 14 (FN14) receptor (See Harada, N. et al, Biochem. Biophys. Res. Commun. 299: 488-93 (2002); Nakayama, M et al, Biochem. Biophys. Res. Communic., 306: 819-825 (2003), a tightly regulated and inducible receptor that has been suggested to signal through a variety of downstream signalling cascades. (Ando, T., et al., Arthritis Res. Ther. 8: R146 (2006); Brown, S. A., et al., Biochem. J. 371: 395-403 (2003); Saitoh, T. et al., J. Biol. Chem. 278: 36005-36012 (2003); Dogra, C. et al., FASEB J. 21: 1857-69 (2007), each of which is incorporated by reference herein).
The term “vascular insufficiency” refers to insufficient blood flow.
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The described invention provides infarct area perfusion-improving pharmaceutical compositions and methods for treatment and repair of an infarct area injury in the aftermath of an acute myocardial infarction resulting from a natural disease process.
In one aspect, the described invention provides a pharmaceutical composition for treating an infarct area injury of a revascularized subject following an acute myocardial infarction resulting from a natural disease process, comprising: (a) an infarct area-improving amount of a sterile isolated chemotactic hematopoietic stem cell product, wherein the infarct area-improving amount of the sterile isolated chemotactic hematopoietic stem cell product comprises an enriched population of at leas 10×106 isolated autologous CD34+ cells containing a subpopulation of potent cells expressing CXCR-4 and having CXCR-4-mediated chemotactic activity; (b) a stabilizing amount of serum, wherein the stabilizing amount of serum is greater than 20% (v/v), and (c) a therapeutically effective amount of at least one compatible agent that promotes cardiomyocyte growth, wherein the composition is administered to the subject parenterally through a catheter; and wherein at least 70% of cells in the enriched population of isolated CD34+ cells containing the subpopulation of potent cells that express CXCR-4 and that have CXCR-4-mediated chemotactic activity when passed through the catheter and when tested in vitro are CD34+ cells, and wherein the enriched population of isolated CD34+ cells containing a subpopulation of potent cells that express CXCR-4 and that have CXCR-4-mediated chemotactic activity when passed through the catheter and tested in vitro (1) retains the CXCR-4-mediated chemotactic activity; (2) is at least about 70% viable; and (3) is able to form hematopoietic colonies in vitro, for at least about 24 hours following acquisition from the subject of the enriched population of CD34+ cells containing the subpopulation of potent cells that express CXCR-4. According to some such embodiments, the agent that promotes cardiomyocyte growth is selected from the group consisting of an angiotensin converting enzyme inhibitor, a beta-blocker, a diuretic, an anti-arrhythmic agent, an anti-anginal agent, a tyrosine kinase receptor agonist, a vasoactive agent, an anticoagulant agent, a fibrinolytic agent, and a hypercholesterolemic agent. According to another embodiment, the at least one compatible agent that promotes cardiomyocyte growth is the tyrosine kinase receptor agonist neuregulin 1. Additional compatible active agents that promote cardiomyocyte growth include, but are not limited to, vascular endothelial growth factor (VEGF)-A, VEGF-B, VEGF-C, VEGF-D, placental growth factor (PIGF), catecholamines, such as, but not limited to, norepinephrine, endothelin-1, a prostaglandin F2α, angiotensin II, phorbol esters, neuropeptide Y, active transforming growth factor β1 (TGF-1β), Gq protein, diacyl glyercol (DAG), salusin-α, salusin-β, insulin-like growth factor (IGF-1), myostatin, granulocyte colony-stimulating factor (G-CSF), macrophage colony-stimulating factor (M-CSF), tumor necrosis factor-like weak inducer of apoptosis (TWEAK), thiazolidinediones, such as, but not limited to, rosiglitazone, and variants or recombinant derivatives thereof.
According to one embodiment, the composition reduces the infarct area injury more than composition components (a) plus (b), or component (c) alone. In some embodiments, the composition improves microvascular blood flow in the infarct area, when compared to controls. In some embodiments, the infarct area injury comprises apoptotic cardiomyocyte loss in the infarct area, when compared to controls. In some embodiments, the infarct area injury comprises ventricular remodeling as measured by a drop in LVEF or an increase in LVESV. In some embodiments, the infarct area injury comprises a progressive decline in heart muscle function resulting from an AMI. In some embodiments, the infarct area injury comprises hypoperfusion in the peri-infarct border zone, relative to controls. In some embodiments, the infarct area injury comprises myocardial hibernation in the peri-infarct border zone, when compared to controls. In some embodiments, the composition increases perfusion of at least one ischemic peri-infarct zone of myocardial tissue, when compared to controls. In some embodiments, the composition increases perfusion to hibernating myocardium in at least one peri-infarct zone of myocardial tissue, when compared to controls. In some embodiments, the composition decreases infarct area, when compared to controls. In some embodiments, the composition decreases infarct mass, when compared to controls.
According to another embodiment, the enriched population of isolated CD34+ cells containing a subpopulation of potent cells expressing CXCR-4 and having CXCR-4-mediated chemotactic activity is purified from cellular components of a bone marrow aspirate acquired from the subject. According to another embodiment, the enriched population of isolated CD34+ cells containing a subpopulation of potent cells expressing CXCR-4 and having CXCR-4-mediated chemotactic activity is purified from peripheral blood. According to one embodiment, the enriched population of CD34+ cells containing a subpopulation of potent cells that express CXCR-4 and that have CXCR-4-mediated chemotactic activity (a) is capable of forming hematopoietic colonies in vitro, and (b) retains at least 2% of the CXCR-4-mediated chemotactic activity, for at least about 48 hours following acquisition from the subject of the enriched population of CD34+ cells containing the subpopulation of potent cells that express CXCR-4 in (a). According to one embodiment, the enriched population of CD34+ cells containing a subpopulation of potent cells that express CXCR-4 and that have CXCR-4-mediated chemotactic activity (a) is capable of forming hematopoietic colonies in and (b) retains at least 2% of the CXCR-4-mediated chemotactic activity, for at least about 72 hours following acquisition from the subject of the enriched population of CD34+ cells containing the subpopulation of potent cells that express CXCR-4 in (a). According to one embodiment, the enriched population of CD34+ cells containing a subpopulation of potent cells that express CXCR-4 and that have CXCR-4-mediated chemotactic activity retains at least 2% of the CXCR-4-mediated chemotactic activity for at least about 24 hours following acquisition from the subject of the enriched population of CD34+ cells containing the subpopulation of potent cells that express CXCR-4 in (a).
According to one embodiment, the chemotactic hematopoietic stem cell product is prepared by isolating or purifying the enriched population of isolated CD34+ cells containing a subpopulation of potent cells expressing CXCR-4 and having CXCR-4-mediated chemotactic activity from bone marrow harvested from the subject. According to another embodiment, the chemotactic hematopoietic stem cell product is prepared by isolating or purifying the enriched population of isolated CD34+ cells containing a subpopulation of potent cells expressing CXCR-4 and having CXCR-4-mediated chemotactic activity from peripheral blood.
According to the described invention, the chemotactic hematopoietic stem cell product enriched for CD34+ cells contains at least about 70% pure CD34+ cells. In some embodiments, the chemotactic hematopoietic stem cell product enriched for CD34+ cells contains at least about 75% pure CD34+ cells. In some embodiments, the chemotactic hematopoietic stem cell product enriched for CD34+ cells contains at least about 80% pure CD34+ cells. In some embodiments, the chemotactic hematopoietic stem cell product enriched for CD34+ cells contains at least about 85% pure CD34+ cells. In some embodiments, the chemotactic hematopoietic stem cell product enriched for CD34+ cells contains at least about 90% pure CD34+ cells. In some embodiments, the chemotactic hematopoietic stem cell product enriched for CD34+ cells contains at least about 95% pure CD34+ cells.
In another embodiment, at least about 70% of the CD34+ cells are viable for at least about 24 hours following acquisition of the enriched population of CD34+ cells. In another embodiment, at least about 75% of the CD34+ cells are viable for at least about 24 hours following acquisition of the enriched population of CD34+ cells. In another embodiment, at least about 80% of the CD34+ cells are viable for at least 24 hours following acquisition of the enriched population of CD34+ cells. In another embodiment, at least about 85% of the CD34+ cells are viable for at least 24 hours following acquisition of the enriched population of CD34+ cells. In some embodiments, at least about 90% of the CD34+ cells are viable for at least 24 hours following acquisition of the enriched population of CD34+ cells. In some embodiments, at least about 95% of the CD34+ cells are viable for at least about 24 following acquisition of the enriched population of CD34+ cells.
In another embodiment, at least about 70% of the CD34+ cells are viable for at least about 48 hours following acquisition of the enriched population of CD34+ cells. In another embodiment, at least about 75% of the CD34+ cells are viable for at least about 48 hours following acquisition of the enriched population of CD34+ cells. In another embodiment, at least about 80% of the CD34+ cells are viable for at least 48 hours following acquisition of the enriched population of CD34+ cells. In another embodiment, at least about 85% of the CD34+ cells are viable for at least 48 hours following acquisition of the enriched population of CD34+ cells. In some embodiments, at least about 90% of the CD34+ cells are viable for at least 48 hours following acquisition of the enriched population of CD34+ cells. In some embodiments, at least about 95% of the CD34+ cells are viable for at least about 48 following acquisition of the enriched population of CD34+ cells.
In another embodiment, at least about 70% of the CD34+ cells are viable for at least about 72 hours following acquisition of the enriched population of CD34+ cells. In another embodiment, at least about 75% of the CD34+ cells are viable for at least about 72 hours following acquisition of the enriched population of CD34+ cells. In another embodiment, at least about 80% of the CD34+ cells are viable for at least 72 hours following acquisition of the enriched population of CD34+ cells. In another embodiment, at least about 85% of the CD34+ cells are viable for at least 72 hours following acquisition of the enriched population of CD34+ cells. In some embodiments, at least about 90% of the CD34+ cells are viable for at least 72 hours following acquisition of the enriched population of CD34+ cells. In some embodiments, at least about 95% of the CD34+ cells are viable for at least about 72 following acquisition of the enriched population of CD34+ cells.
In another embodiment, the CD34+ cells can form hematopoietic colonies in vitro for at least about 24 hours following acquisition of the enriched population of CD34+ cells. In another embodiment, the CD34+ cells can form hematopoietic colonies in vitro for at least about 48 hours following acquisition of the enriched population of CD34+ cells. In another embodiment, the CD34+ cells can form hematopoietic colonies in vitro for at least about 72 hours following acquisition of the enriched population of CD34+ cells.
According to another embodiment, the infarct area perfusion-improving composition comprises at least about 10 million CD34+ cells acquired from the subject and containing a subpopulation of potent cells expressing CXCR-4 and having CXCR-4-mediated chemotactic activity. According to another embodiment, the infarct area perfusion-improving composition further comprises at least about 11 million CD34+ cells acquired from the subject and containing a subpopulation of potent cells expressing CXCR-4 and having CXCR-4-mediated chemotactic activity. According to another embodiment, the infarct area perfusion-improving composition further comprises at least about 12 million CD34+ cells acquired from the subject and containing a subpopulation of potent cells expressing CXCR-4 and having CXCR-4-mediated chemotactic activity. According to another embodiment, the infarct area perfusion-improving composition further comprises at least about 13 million CD34+ cells acquired from the subject and containing a subpopulation of potent cells expressing CXCR-4 and having CXCR-4-mediated chemotactic activity. According to another embodiment, the infarct area perfusion-improving composition further comprises at least about 14 million CD34+ cells acquired from the subject and containing a subpopulation of potent cells expressing CXCR-4 and having CXCR-4-mediated chemotactic activity. According to another embodiment, the infarct area perfusion-improving composition further comprises at least about 15 million CD34+ cells acquired from the subject and containing a subpopulation of potent cells expressing CXCR-4 and having CXCR-4-mediated chemotactic activity. According to another embodiment, the infarct area perfusion-improving composition further comprises at least about 16 million CD34+ cells acquired from the subject and containing a subpopulation of potent cells expressing CXCR-4 and having CXCR-4-mediated chemotactic activity. According to another embodiment, the infarct area perfusion-improving composition further comprises at least about 17 million CD34+ cells acquired from the subject and containing a subpopulation of potent cells expressing CXCR-4 and having CXCR-4-mediated chemotactic activity. According to another embodiment, the infarct area perfusion-improving composition further comprises at least about 18 million CD34+ cells acquired from the subject and containing a subpopulation of potent cells expressing CXCR-4 and having CXCR-4-mediated chemotactic activity. According to another embodiment, the infarct area perfusion-improving composition further comprises at least about 19 million CD34+ cells acquired from the subject and containing a subpopulation of potent cells expressing CXCR-4 and having CXCR-4-mediated chemotactic activity. According to another embodiment, the infarct area perfusion-improving composition further comprises at least about 20 million CD34+ cells acquired from the subject and containing a subpopulation of potent cells expressing CXCR-4 and having CXCR-4-mediated chemotactic activity. According to another embodiment, the infarct area perfusion-improving composition further comprises at least about 25 million CD34+ cells acquired from the subject and containing a subpopulation of potent cells expressing CXCR-4 and having CXCR-4-mediated chemotactic activity. According to another embodiment, the infarct area perfusion-improving composition further comprises at least about 30 million CD34+ cells acquired from the subject and containing a subpopulation of potent cells expressing CXCR-4 and having CXCR-4-mediated chemotactic activity. According to another embodiment, the infarct area perfusion-improving composition further comprises at least about 35 million CD34+ cells acquired from the subject and containing a subpopulation of potent cells expressing CXCR-4 and having CXCR-4-mediated chemotactic activity. According to another embodiment, the infarct area perfusion-improving composition further comprises at least about 40 million CD34+ cells acquired from the subject and containing a subpopulation of potent cells expressing CXCR-4 and having CXCR-4-mediated chemotactic activity. According to another embodiment, the infarct area perfusion-improving composition further comprises at least about 45 million CD34+ cells acquired from the subject and containing a subpopulation of potent cells expressing CXCR-4 and having CXCR-4-mediated chemotactic activity. According to another embodiment, the infarct area perfusion-improving composition further comprises at least about 50 million CD34+ cells acquired from the subject and containing a subpopulation of potent cells expressing CXCR-4 and having CXCR-4-mediated chemotactic activity. According to another embodiment, the infarct area perfusion-improving composition further comprises at least about 55 million CD34+ cells acquired from the subject and containing a subpopulation of potent cells expressing CXCR-4 and having CXCR-4-mediated chemotactic activity. According to another embodiment, the infarct area perfusion-improving composition further comprises at least about 60 million CD34+ cells acquired from the subject and containing a subpopulation of potent cells expressing CXCR-4 and having CXCR-4-mediated chemotactic activity.
CD34+ cells may be enriched/selected by any techniques known to the skilled artisan. For example, in some embodiments, the population of bone marrow cells comprising CD34+ cells is enriched for cells expressing CD34 cell antigen and CXCR4 cell antigen by fluorescence activated cell sorting (FACS). In some embodiments, CD34+ cells in the bone marrow are enriched/selected by positive or negative immunoseparation techniques. In some embodiments, isolation and/or purification of hematopoietic stem cells from the bone marrow is based on cell fractionation methods based on size and cell density, efflux of metabolic dyes, or resistance to cytotoxic agents. In one embodiment, for example, CD34+ cells in the bone marrow are enriched/selected using a monoclonal anti-CD34 antibody and an immunomagnetic separation technique.
The selected CD34+ cells may be identified, quantified and characterized by techniques known in the art. For example, in some embodiments, the percentage of CD34+ cells in the bone marrow and in the chemotactic hematopoietic stem cell product can be determined by FACS analysis. In another embodiment, CD34 protein expression is quantified by Western blot. The term “Western blot” refers to a method for identifying proteins in a complex mixture; proteins are separated electrophoretically in a gel medium; transferred from the gel to a protein binding sheet or membrane; and the sheet or membrane containing the separated proteins exposed to specific antibodies which bind to, locate, and enable visualization of protein(s) of interest. For example, monoclonal anti-CD34 antibody can be used to detect CD34 protein adhered to a membrane in situ.
In another embodiment, the expression of CD34 mRNA and DNA in the isolated CD34+ cells may be quantified. The term “Northern blot” as used herein refers to a technique in which RNA from a specimen is separated into its component parts on a gel by electrophoresis and transferred to a specifically modified paper support so that the mRNA is fixed in its electrophoretic positions. CD34 related sequences are identified using probes comprising a reporter molecule, such as, without limitation, a radioactive label. In another embodiment, the level of CD34 and/or CXCR4 expression is/are determined by quantitative or semi-quantitative PCR or real time PCR (“RT-PCR”) techniques. The abbreviation “PCR” refers to polymerase chain reaction, which is a technique for amplifying the quantity of DNA, thus making the DNA easier to isolate, clone and sequence. See, e.g., U.S. Pat. Nos. 5,656,493, 5,333,675, 5,234,824, and 5,187,083, each of which is incorporated herein by reference. Real-time PCR is a method of simultaneous DNA quantification and amplification, whereby DNA is specifically amplified by polymerase chain reaction (PCR), and after each round of amplification, the DNA is quantified.
The selected CD34+ hematopoietic stem cells of the chemotactic hematopoietic stem cell product of the described invention contain a subpopulation of CD34+ cells expressing CXCR-4 and having CXCR-4 mediated chemotactic activity. In one embodiment, the hematopoietic stem cell product of the described invention comprises a minimum number of isolated CD34+ hematopoietic stem cells such that a subpopulation of at least 0.5×106 CD34+ cells expressing CXCR-4 and having CXCR-4 mediated chemotactic activity is present. In another embodiment, at least about 2% of the CXCR-4 mediated chemotactic activity of the CD34+CXCR-4+ cells is retained for at least 24 hours following acquisition of the enriched population of CD34+ cells. In another embodiment, at least about 3% of the CXCR-4 mediated chemotactic activity of the CD34+CXCR-4+ cells is retained for at least 24 hours following acquisition of the enriched population of CD34+ cells. In another embodiment, at least about 4% of the CXCR-4 mediated chemotactic activity of the CD34+CXCR-4+ cells is retained for at least 24 hours following acquisition of the enriched population of CD34+ cells. In another embodiment, at least about 5% of the CXCR-4 mediated chemotactic activity of the CD34+CXCR-4+ cells is retained for at least 24 hours following acquisition of the enriched population of CD34+ cells. In another embodiment, at least about 6% of the CXCR-4 mediated chemotactic activity of the CD34+CXCR-4+ cells is retained for at least 24 hours following acquisition of the enriched population of CD34+ cells. In another embodiment, at least about 7% of the CXCR-4 mediated chemotactic activity of the CD34+CXCR-4+ cells is retained for at least 24 hours following acquisition of the enriched population of CD34+ cells. In another embodiment, at least about 8% of the CXCR-4 mediated chemotactic activity of the CD34+CXCR-4+ cells is retained for at least 24 hours following acquisition of the enriched population of CD34+ cells. In another embodiment, at least about 9% of the CXCR-4 mediated chemotactic activity of the CD34+CXCR-4+ cells is retained for at least 24 hours following acquisition of the enriched population of CD34+ cells. In another embodiment, at least about 10% of the CXCR-4 mediated chemotactic activity of the CD34+CXCR-4+ cells is retained for at least 24 hours following acquisition of the enriched population of CD34+ cells. In another embodiment, at least about 11% of the CXCR-4 mediated chemotactic activity of the CD34+CXCR-4+ cells is retained for at least 24 hours following acquisition of the enriched population of CD34+ cells. In another embodiment, at least about 12% of the CXCR-4 mediated chemotactic activity of the CD34+CXCR-4+ cells is retained for at least 24 hours following acquisition of the enriched population of CD34+ cells. In another embodiment, at least about 13% of the CXCR-4 mediated chemotactic activity of the CD34+CXCR-4+ cells is retained for at least 24 hours following acquisition of the enriched population of CD34+ cells. In another embodiment, at least about 14% of the CXCR-4 mediated chemotactic activity of the CD34+CXCR-4+ cells is retained for at least 24 hours following acquisition of the enriched population of CD34+ cells. In another embodiment, at least about 15% of the CXCR-4 mediated chemotactic activity of the CD34+CXCR-4+ cells is retained for at least 24 hours following acquisition of the enriched population of CD34+ cells. In another embodiment, at least about 16% of the CXCR-4 mediated chemotactic activity of the CD34+CXCR-4+ cells is retained for at least 24 hours following acquisition of the enriched population of CD34+ cells. In another embodiment, at least about 17% of the CXCR-4 mediated chemotactic activity of the CD34+CXCR-4+ cells is retained for at least 24 hours following acquisition of the enriched population of CD34+ cells. In another embodiment, at least about 18% of the CXCR-4 mediated chemotactic activity of the CD34+CXCR-4+ cells is retained for at least 24 hours following acquisition of the enriched population of CD34+ cells. In another embodiment, at least about 19% of the CXCR-4 mediated chemotactic activity of the CD34+CXCR-4+ cells is retained for at least 24 hours following acquisition of the enriched population of CD34+ cells. In another embodiment, at least about 20% of the CXCR-4 mediated chemotactic activity of the CD34+CXCR-4+ cells is retained for at least 24 hours following acquisition of the enriched population of CD34+ cells. In another embodiment, at least about 21% of the CXCR-4 mediated chemotactic activity of the CD34+CXCR-4+ cells is retained for at least 24 hours following acquisition of the enriched population of CD34+ cells. In another embodiment, at least about 22% of the CXCR-4 mediated chemotactic activity of the CD34+CXCR-4+ cells is retained for at least 24 hours following acquisition of the enriched population of CD34+ cells. In another embodiment, at least about 23% of the CXCR-4 mediated chemotactic activity of the CD34+CXCR-4+ cells is retained for at least 24 hours following acquisition of the enriched population of CD34+ cells. In another embodiment, at least about 24% of the CXCR-4 mediated chemotactic activity of the CD34+CXCR-4+ cells is retained for at least 24 hours following acquisition of the enriched population of CD34+ cells. In another embodiment, at least about 25% of the CXCR-4 mediated chemotactic activity of the CD34+CXCR-4+ cells is retained for at least 24 hours following acquisition of the enriched population of CD34+ cells. In another embodiment, at least about 26% of the CXCR-4 mediated chemotactic activity of the CD34+CXCR-4+ cells is retained for at least 24 hours following acquisition of the enriched population of CD34+ cells. In another embodiment, at least about 27% of the CXCR-4 mediated chemotactic activity of the CD34+CXCR-4+ cells is retained for at least 24 hours following acquisition of the enriched population of CD34+ cells. In another embodiment, at least about 28% of the CXCR-4 mediated chemotactic activity of the CD34+CXCR-4+ cells is retained for at least 24 hours following acquisition of the enriched population of CD34+ cells. In another embodiment, at least about 29% of the CXCR-4 mediated chemotactic activity of the CD34+CXCR-4+ cells is retained for at least 24 hours following acquisition of the enriched population of CD34+ cells. In another embodiment, at least about 30% of the CXCR-4 mediated chemotactic activity of the CD34+CXCR-4+ cells is retained for at least 24 hours following acquisition of the enriched population of CD34+ cells. In another embodiment, at least about 31% of the CXCR-4 mediated chemotactic activity of the CD34+CXCR-4+ cells is retained for at least 24 hours following acquisition of the enriched population of CD34+ cells. In another embodiment, at least about 32% of the CXCR-4 mediated chemotactic activity of the CD34+CXCR-4+ cells is retained for at least 24 hours following acquisition of the enriched population of CD34+ cells. In another embodiment, at least about 33% of the CXCR-4 mediated chemotactic activity of the CD34+CXCR-4+ cells is retained for at least 24 hours following acquisition of the enriched population of CD34+ cells. In another embodiment, at least about 34% of the CXCR-4 mediated chemotactic activity of the CD34+CXCR-4+ cells is retained for at least 24 hours following acquisition of the enriched population of CD34+ cells. In another embodiment, at least about 35% of the CXCR-4 mediated chemotactic activity of the CD34+CXCR-4+ cells is retained for at least 24 hours following acquisition of the enriched population of CD34+ cells. In another embodiment, at least about 35% of the CXCR-4 mediated chemotactic activity of the CD34+CXCR-4+ cells is retained for at least 24 hours following acquisition of the enriched population of CD34+ cells. In another embodiment, at least about 40% of the CXCR-4 mediated chemotactic activity of the CD34+CXCR-4+ cells is retained for at least 24 hours following acquisition of the enriched population of CD34+ cells. In another embodiment, at least about 45% of the CXCR-4 mediated chemotactic activity of the CD34+CXCR-4+ cells is retained for at least 24 hours following acquisition of the enriched population of CD34+ cells. In another embodiment, at least about 50% of the CXCR-4 mediated chemotactic activity of the CD34+CXCR-4+ cells is retained for at least 24 hours following acquisition of the enriched population of CD34+ cells. In another embodiment, at least about 55% of the CXCR-4 mediated chemotactic activity of the CD34+CXCR-4+ cells is retained for at least 24 hours following acquisition of the enriched population of CD34+ cells. In another embodiment, at least about 60% of the CXCR-4 mediated chemotactic activity of the CD34+CXCR-4+ cells is retained for at least 24 hours following acquisition of the enriched population of CD34+ cells. In another embodiment, at least about 65% of the CXCR-4 mediated chemotactic activity of the CD34+CXCR-4+ cells is retained for at least 24 hours following acquisition of the enriched population of CD34+ cells. In another embodiment, at least about 70% of the CXCR-4 mediated chemotactic activity of the CD34+CXCR-4+ cells is retained for at least 24 hours following acquisition of the enriched population of CD34+ cells. In another embodiment, at least about 75% of the CXCR-4 mediated chemotactic activity of the CD34+CXCR-4+ cells is retained for at least 24 hours following acquisition of the enriched population of CD34+ cells. In another embodiment, at least about 80% of the CXCR-4 mediated chemotactic activity of the CD34+CXCR-4+ cells is retained for at least 24 hours following acquisition of the enriched population of CD34+ cells. In another embodiment, at least about 85% of the CXCR-4 mediated chemotactic activity of the CD34+CXCR-4+ cells is retained for at least 24 hours following acquisition of the enriched population of CD34+ cells. In another embodiment, at least about 90% of the CXCR-4 mediated chemotactic activity of the CD34+CXCR-4+ cells is retained for at least 24 hours following acquisition of the enriched population of CD34+ cells. In another embodiment, at least about 95% of the CXCR-4 mediated chemotactic activity of the CD34+CXCR-4+ cells is retained for at least 24 hours following acquisition of the enriched population of CD34+ cells.
In another embodiment, at least about 2% of the CXCR-4 mediated chemotactic activity of the CD34+CXCR-4+ cells is retained for at least 48 hours following acquisition of the enriched population of CD34+ cells. In another embodiment, at least about 3% of the CXCR-4 mediated chemotactic activity of the CD34+CXCR-4+ cells is retained for at least 48 hours following acquisition of the enriched population of CD34+ cells. In another embodiment, at least about 4% of the CXCR-4 mediated chemotactic activity of the CD34+CXCR-4+ cells is retained for at least 48 hours following acquisition of the enriched population of CD34+ cells. In another embodiment, at least about 5% of the CXCR-4 mediated chemotactic activity of the CD34+CXCR-4+ cells is retained for at least 48 hours following acquisition of the enriched population of CD34+ cells. In another embodiment, at least about 6% of the CXCR-4 mediated chemotactic activity of the CD34+CXCR-4+ cells is retained for at least 48 hours following acquisition of the enriched population of CD34+ cells. In another embodiment, at least about 7% of the CXCR-4 mediated chemotactic activity of the CD34+CXCR-4+ cells is retained for at least 48 hours following acquisition of the enriched population of CD34+ cells. In another embodiment, at least about 8% of the CXCR-4 mediated chemotactic activity of the CD34+CXCR-4+ cells is retained for at least 48 hours following acquisition of the enriched population of CD34+ cells. In another embodiment, at least about 9% of the CXCR-4 mediated chemotactic activity of the CD34+CXCR-4+ cells is retained for at least 48 hours following acquisition of the enriched population of CD34+ cells. In another embodiment, at least about 10% of the CXCR-4 mediated chemotactic activity of the CD34+CXCR-4+ cells is retained for at least 48 hours following acquisition of the enriched population of CD34+ cells. In another embodiment, at least about 11% of the CXCR-4 mediated chemotactic activity of the CD34+CXCR-4+ cells is retained for at least 48 hours following acquisition of the enriched population of CD34+ cells. In another embodiment, at least about 12% of the CXCR-4 mediated chemotactic activity of the CD34+CXCR-4+ cells is retained for at least 48 hours following acquisition of the enriched population of CD34+ cells. In another embodiment, at least about 13% of the CXCR-4 mediated chemotactic activity of the CD34+CXCR-4+ cells is retained for at least 48 hours following acquisition of the enriched population of CD34+ cells. In another embodiment, at least about 14% of the CXCR-4 mediated chemotactic activity of the CD34+CXCR-4+ cells is retained for at least 48 hours following acquisition of the enriched population of CD34+ cells. In another embodiment, at least about 15% of the CXCR-4 mediated chemotactic activity of the CD34+CXCR-4+ cells is retained for at least 48 hours following acquisition of the enriched population of CD34+ cells. In another embodiment, at least about 16% of the CXCR-4 mediated chemotactic activity of the CD34+CXCR-4+ cells is retained for at least 48 hours following acquisition of the enriched population of CD34+ cells. In another embodiment, at least about 17% of the CXCR-4 mediated chemotactic activity of the CD34+CXCR-4+ cells is retained for at least 48 hours following acquisition of the enriched population of CD34+ cells. In another embodiment, at least about 18% of the CXCR-4 mediated chemotactic activity of the CD34+CXCR-4+ cells is retained for at least 48 hours following acquisition of the enriched population of CD34+ cells. In another embodiment, at least about 19% of the CXCR-4 mediated chemotactic activity of the CD34+CXCR-4+ cells is retained for at least 48 hours following acquisition of the enriched population of CD34+ cells. In another embodiment, at least about 20% of the CXCR-4 mediated chemotactic activity of the CD34+CXCR-4+ cells is retained for at least 48 hours following acquisition of the enriched population of CD34+ cells. In another embodiment, at least about 21% of the CXCR-4 mediated chemotactic activity of the CD34+CXCR-4+ cells is retained for at least 48 hours following acquisition of the enriched population of CD34+ cells. In another embodiment, at least about 22% of the CXCR-4 mediated chemotactic activity of the CD34+CXCR-4+ cells is retained for at least 48 hours following acquisition of the enriched population of CD34+ cells. In another embodiment, at least about 23% of the CXCR-4 mediated chemotactic activity of the CD34+CXCR-4+ cells is retained for at least 48 hours following acquisition of the enriched population of CD34+ cells. In another embodiment, at least about 24% of the CXCR-4 mediated chemotactic activity of the CD34+CXCR-4+ cells is retained for at least 48 hours following acquisition of the enriched population of CD34+ cells. In another embodiment, at least about 25% of the CXCR-4 mediated chemotactic activity of the CD34+CXCR-4+ cells is retained for at least 48 hours following acquisition of the enriched population of CD34+ cells. In another embodiment, at least about 26% of the CXCR-4 mediated chemotactic activity of the CD34+CXCR-4+ cells is retained for at least 48 hours following acquisition of the enriched population of CD34+ cells. In another embodiment, at least about 27% of the CXCR-4 mediated chemotactic activity of the CD34+CXCR-4+ cells is retained for at least 48 hours following acquisition of the enriched population of CD34+ cells. In another embodiment, at least about 28% of the CXCR-4 mediated chemotactic activity of the CD34+CXCR-4+ cells is retained for at least 48 hours following acquisition of the enriched population of CD34+ cells. In another embodiment, at least about 29% of the CXCR-4 mediated chemotactic activity of the CD34+CXCR-4+ cells is retained for at least 48 hours following acquisition of the enriched population of CD34+ cells. In another embodiment, at least about 30% of the CXCR-4 mediated chemotactic activity of the CD34+CXCR-4+ cells is retained for at least 48 hours following acquisition of the enriched population of CD34+ cells. In another embodiment, at least about 31% of the CXCR-4 mediated chemotactic activity of the CD34+CXCR-4+ cells is retained for at least 48 hours following acquisition of the enriched population of CD34+ cells. In another embodiment, at least about 32% of the CXCR-4 mediated chemotactic activity of the CD34+CXCR-4+ cells is retained for at least 48 hours following acquisition of the enriched population of CD34+ cells. In another embodiment, at least about 33% of the CXCR-4 mediated chemotactic activity of the CD34+CXCR-4+ cells is retained for at least 48 hours following acquisition of the enriched population of CD34+ cells. In another embodiment, at least about 34% of the CXCR-4 mediated chemotactic activity of the CD34+CXCR-4+ cells is retained for at least 48 hours following acquisition of the enriched population of CD34+ cells. In another embodiment, at least about 35% of the CXCR-4 mediated chemotactic activity of the CD34+CXCR-4+ cells is retained for at least 48 hours following acquisition of the enriched population of CD34+ cells. In another embodiment, at least about 40% of the CXCR-4 mediated chemotactic activity of the CD34+CXCR-4+ cells is retained for at least 48 hours following acquisition of the enriched population of CD34+ cells. In another embodiment, at least about 45% of the CXCR-4 mediated chemotactic activity of the CD34+CXCR-4+ cells is retained for at least 48 hours following acquisition of the enriched population of CD34+ cells. In another embodiment, at least about 50% of the CXCR-4 mediated chemotactic activity of the CD34+CXCR-4+ cells is retained for at least 48 hours following acquisition of the enriched population of CD34+ cells. In another embodiment, at least about 55% of the CXCR-4 mediated chemotactic activity of the CD34+CXCR-4+ cells is retained for at least 48 hours following acquisition of the enriched population of CD34+ cells. In another embodiment, at least about 60% of the CXCR-4 mediated chemotactic activity of the CD34+CXCR-4+ cells is retained for at least 48 hours following acquisition of the enriched population of CD34+ cells. In another embodiment, at least about 65% of the CXCR-4 mediated chemotactic activity of the CD34+CXCR-4+ cells is retained for at least 48 hours following acquisition of the enriched population of CD34+ cells. In another embodiment, at least about 70% of the CXCR-4 mediated chemotactic activity of the CD34+CXCR-4+ cells is retained for at least 48 hours following acquisition of the enriched population of CD34+ cells. In another embodiment, at least about 75% of the CXCR-4 mediated chemotactic activity of the CD34+CXCR-4+ cells is retained for at least 48 hours following acquisition of the enriched population of CD34+ cells. In another embodiment, at least about 80% of the CXCR-4 mediated chemotactic activity of the CD34+CXCR-4+ cells is retained for at least 48 hours following acquisition of the enriched population of CD34+ cells. In another embodiment, at least about 85% of the CXCR-4 mediated chemotactic activity of the CD34+CXCR-4+ cells is retained for at least 48 hours following acquisition of the enriched population of CD34+ cells. In another embodiment, at least about 90% of the CXCR-4 mediated chemotactic activity of the CD34+CXCR-4+ cells is retained for at least 48 hours following acquisition of the enriched population of CD34+ cells. In another embodiment, at least about 95% of the CXCR-4 mediated chemotactic activity of the CD34+CXCR-4+ cells is retained for at least 48 hours following acquisition of the enriched population of CD34+ cells.
In another embodiment, at least about 2% of the CXCR-4 mediated chemotactic activity of the CD34+CXCR-4+ cells is retained for at least 72 hours following acquisition of the enriched population of CD34+ cells. In another embodiment, at least about 3% of the CXCR-4 mediated chemotactic activity of the CD34+CXCR-4+ cells is retained for at least 72 hours following acquisition of the enriched population of CD34+ cells. In another embodiment, at least about 4% of the CXCR-4 mediated chemotactic activity of the CD34+CXCR-4+ cells is retained for at least 72 hours following acquisition of the enriched population of CD34+ cells. In another embodiment, at least about 5% of the CXCR-4 mediated chemotactic activity of the CD34+CXCR-4+ cells is retained for at least 72 hours following acquisition of the enriched population of CD34+ cells. In another embodiment, at least about 6% of the CXCR-4 mediated chemotactic activity of the CD34+CXCR-4+ cells is retained for at least 72 hours following acquisition of the enriched population of CD34+ cells. In another embodiment, at least about 7% of the CXCR-4 mediated chemotactic activity of the CD34+CXCR-4+ cells is retained for at least 72 hours following acquisition of the enriched population of CD34+ cells. In another embodiment, at least about 8% of the CXCR-4 mediated chemotactic activity of the CD34+CXCR-4+ cells is retained for at least 72 hours following acquisition of the enriched population of CD34+ cells. In another embodiment, at least about 9% of the CXCR-4 mediated chemotactic activity of the CD34+CXCR-4+ cells is retained for at least 72 hours following acquisition of the enriched population of CD34+ cells. In another embodiment, at least about 10% of the CXCR-4 mediated chemotactic activity of the CD34+CXCR-4+ cells is retained for at least 72 hours following acquisition of the enriched population of CD34+ cells. In another embodiment, at least about 11% of the CXCR-4 mediated chemotactic activity of the CD34+CXCR-4+ cells is retained for at least 72 hours following acquisition of the enriched population of CD34+ cells. In another embodiment, at least about 12% of the CXCR-4 mediated chemotactic activity of the CD34+CXCR-4+ cells is retained for at least 72 hours following acquisition of the enriched population of CD34+ cells. In another embodiment, at least about 13% of the CXCR-4 mediated chemotactic activity of the CD34+CXCR-4+ cells is retained for at least 72 hours following acquisition of the enriched population of CD34+ cells. In another embodiment, at least about 14% of the CXCR-4 mediated chemotactic activity of the CD34+CXCR-4+ cells is retained for at least 72 hours following acquisition of the enriched population of CD34+ cells. In another embodiment, at least about 15% of the CXCR-4 mediated chemotactic activity of the CD34+CXCR-4+ cells is retained for at least 72 hours following acquisition of the enriched population of CD34+ cells. In another embodiment, at least about 16% of the CXCR-4 mediated chemotactic activity of the CD34+CXCR-4+ cells is retained for at least 72 hours following acquisition of the enriched population of CD34+ cells. In another embodiment, at least about 17% of the CXCR-4 mediated chemotactic activity of the CD34+CXCR-4+ cells is retained for at least 72 hours following acquisition of the enriched population of CD34+ cells. In another embodiment, at least about 18% of the CXCR-4 mediated chemotactic activity of the CD34+CXCR-4+ cells is retained for at least 72 hours following acquisition of the enriched population of CD34+ cells. In another embodiment, at least about 19% of the CXCR-4 mediated chemotactic activity of the CD34+CXCR-4+ cells is retained for at least 72 hours following acquisition of the enriched population of CD34+ cells. In another embodiment, at least about 20% of the CXCR-4 mediated chemotactic activity of the CD34+CXCR-4+ cells is retained for at least 72 hours following acquisition of the enriched population of CD34+ cells. In another embodiment, at least about 21% of the CXCR-4 mediated chemotactic activity of the CD34+CXCR-4+ cells is retained for at least 72 hours following acquisition of the enriched population of CD34+ cells. In another embodiment, at least about 22% of the CXCR-4 mediated chemotactic activity of the CD34+CXCR-4+ cells is retained for at least 72 hours following acquisition of the enriched population of CD34+ cells. In another embodiment, at least about 23% of the CXCR-4 mediated chemotactic activity of the CD34+CXCR-4+ cells is retained for at least 72 hours following acquisition of the enriched population of CD34+ cells. In another embodiment, at least about 24% of the CXCR-4 mediated chemotactic activity of the CD34+CXCR-4+ cells is retained for at least 72 hours following acquisition of the enriched population of CD34+ cells. In another embodiment, at least about 25% of the CXCR-4 mediated chemotactic activity of the CD34+CXCR-4+ cells is retained for at least 72 hours following acquisition of the enriched population of CD34+ cells. In another embodiment, at least about 26% of the CXCR-4 mediated chemotactic activity of the CD34+CXCR-4+ cells is retained for at least 72 hours following acquisition of the enriched population of CD34+ cells. In another embodiment, at least about 27% of the CXCR-4 mediated chemotactic activity of the CD34+CXCR-4+ cells is retained for at least 72 hours following acquisition of the enriched population of CD34+ cells. In another embodiment, at least about 28% of the CXCR-4 mediated chemotactic activity of the CD34+CXCR-4+ cells is retained for at least 72 hours following acquisition of the enriched population of CD34+ cells. In another embodiment, at least about 29% of the CXCR-4 mediated chemotactic activity of the CD34+CXCR-4+ cells is retained for at least 72 hours following acquisition of the enriched population of CD34+ cells. In another embodiment, at least about 30% of the CXCR-4 mediated chemotactic activity of the CD34+CXCR-4+ cells is retained for at least 72 hours following acquisition of the enriched population of CD34+ cells. In another embodiment, at least about 31% of the CXCR-4 mediated chemotactic activity of the CD34+CXCR-4+ cells is retained for at least 72 hours following acquisition of the enriched population of CD34+ cells. In another embodiment, at least about 32% of the CXCR-4 mediated chemotactic activity of the CD34+CXCR-4+ cells is retained for at least 72 hours following acquisition of the enriched population of CD34+ cells. In another embodiment, at least about 33% of the CXCR-4 mediated chemotactic activity of the CD34+CXCR-4+ cells is retained for at least 72 hours following acquisition of the enriched population of CD34+ cells. In another embodiment, at least about 34% of the CXCR-4 mediated chemotactic activity of the CD34+CXCR-4+ cells is retained for at least 72 hours following acquisition of the enriched population of CD34+ cells. In another embodiment, at least about 35% of the CXCR-4 mediated chemotactic activity of the CD34+CXCR-4+ cells is retained for at least 72 hours following acquisition of the enriched population of CD34+ cells. In another embodiment, at least about 40% of the CXCR-4 mediated chemotactic activity of the CD34+CXCR-4+ cells is retained for at least 72 hours following acquisition of the enriched population of CD34+ cells. In another embodiment, at least about 45% of the CXCR-4 mediated chemotactic activity of the CD34+CXCR-4+ cells is retained for at least 72 hours following acquisition of the enriched population of CD34+ cells. In another embodiment, at least about 50% of the CXCR-4 mediated chemotactic activity of the CD34+CXCR-4+ cells is retained for at least 72 hours following acquisition of the enriched population of CD34+ cells. In another embodiment, at least about 55% of the CXCR-4 mediated chemotactic activity of the CD34+CXCR-4+ cells is retained for at least 72 hours following acquisition of the enriched population of CD34+ cells. In another embodiment, at least about 60% of the CXCR-4 mediated chemotactic activity of the CD34+CXCR-4+ cells is retained for at least 72 hours following acquisition of the enriched population of CD34+ cells. In another embodiment, at least about 65% of the CXCR-4 mediated chemotactic activity of the CD34+CXCR-4+ cells is retained for at least 72 hours following acquisition of the enriched population of CD34+ cells. In another embodiment, at least about 70% of the CXCR-4 mediated chemotactic activity of the CD34+CXCR-4+ cells is retained for at least 72 hours following acquisition of the enriched population of CD34+ cells. In another embodiment, at least about 75% of the CXCR-4 mediated chemotactic activity of the CD34+CXCR-4+ cells is retained for at least 72 hours following acquisition of the enriched population of CD34+ cells. In another embodiment, at least about 80% of the CXCR-4 mediated chemotactic activity of the CD34+CXCR-4+ cells is retained for at least 72 hours following acquisition of the enriched population of CD34+ cells. In another embodiment, at least about 85% of the CXCR-4 mediated chemotactic activity of the CD34+CXCR-4+ cells is retained for at least 72 hours following acquisition of the enriched population of CD34+ cells. In another embodiment, at least about 90% of the CXCR-4 mediated chemotactic activity of the CD34+CXCR-4+ cells is retained for at least 72 hours following acquisition of the enriched population of CD34+ cells. In another embodiment, at least about 95% of the CXCR-4 mediated chemotactic activity of the CD34+CXCR-4+ cells is retained for at least 72 hours following acquisition of the enriched population of CD34+ cells.
In another embodiment, at least an average of about 17% of the CXCR-4 mediated chemotactic activity of the CD34+CXCR-4+ cells is retained for at least 24 hours following acquisition of the enriched population of CD34+ cells. In another embodiment, at least an average of about 17% of the CXCR-4 mediated chemotactic activity of the CD34+CXCR-4+ cells is retained for at least 48 hours following acquisition of the enriched population of CD34+ cells. In another embodiment, at least an average of about 17% of the CXCR-4 mediated chemotactic activity of the CD34+CXCR-4+ cells is retained for at least 72 hours following acquisition of the enriched population of CD34+ cells. In another embodiment, the CD34+CXCR-4+ cells in the chemotactic hematopoietic cell product retain at least about 2% of the CXCR-4 mediated chemotactic activity for at least 72 hours following acquisition of the enriched population of CD34+ cells.
The pharmaceutical composition of the invention further comprises serum at a concentration of at least 10% by volume of the infarct area perfusion-improving composition. In one embodiment, the serum is autologous. In another embodiment, the serum is a synthetic or recombinant serum. In another embodiment, the minimum concentration of serum present in the infarct area perfusion-improving composition is at least about 10% expressed as ml/100 cc final volume of the composition. In another embodiment, the minimum concentration of serum present in the infarct area perfusion-improving composition is at least about 15% expressed as ml/100 cc final volume of the composition. In another embodiment, the minimum concentration of serum present in the infarct area perfusion-improving composition is at least about 20% expressed as ml/100 cc final volume of the composition. In another embodiment, the minimum concentration of serum present in the infarct area perfusion-improving composition is at least about 21% expressed as ml/100 cc final volume of the composition. In another embodiment, the minimum concentration of serum present in the infarct area perfusion-improving composition is at least about 22% expressed as ml/100 cc final volume of the composition. In another embodiment, the minimum concentration of serum present in the infarct area perfusion-improving composition is at least about 23% expressed as ml/100 cc final volume of the composition. In another embodiment, the minimum concentration of serum present in the infarct area perfusion-improving composition is at least about 24% expressed as ml/100 cc final volume of the composition. In another embodiment, the minimum concentration of serum present in the infarct area perfusion-improving composition is at least about 25% expressed as ml/100 cc final volume of the composition. In another embodiment, the minimum concentration of serum present in the infarct area perfusion-improving composition is at least about 26% expressed as ml/100 cc final volume of the composition. In another embodiment, the minimum concentration of serum present in the infarct area perfusion-improving composition is at least about 27% expressed as ml/100 cc final volume of the composition. In another embodiment, the minimum concentration of serum present in the infarct area perfusion-improving composition is at least about 28% expressed as ml/100 cc final volume of the composition. In another embodiment, the minimum concentration of serum present in the infarct area perfusion-improving composition is at least about 29% expressed as ml/100 cc final volume of the composition. In another embodiment, the minimum concentration of serum present in the infarct area perfusion-improving composition is at least about 30% expressed as ml/100 cc final volume of the composition. In another embodiment, the minimum concentration of serum present in the infarct area perfusion-improving composition is at least about 31% expressed as ml/100 cc final volume of the composition. In another embodiment, the minimum concentration of serum present in the infarct area perfusion-improving composition is at least about 32% expressed as ml/100 cc final volume of the composition. In another embodiment, the minimum concentration of serum present in the infarct area perfusion-improving composition is at least about 33% expressed as ml/100 cc final volume of the composition. In another embodiment, the minimum concentration of serum present in the infarct area perfusion-improving composition is at least about 34% expressed as ml/100 cc final volume of the composition. In another embodiment, the minimum concentration of serum present in the infarct area perfusion-improving composition is at least about 35% expressed as ml/100 cc final volume of the composition. In another embodiment, the minimum concentration of serum present in the infarct area perfusion-improving composition is at least about 36% expressed as ml/100 cc final volume of the composition. In another embodiment, the minimum concentration of serum present in the infarct area perfusion-improving composition is at least about 37% expressed as ml/100 cc final volume of the composition. In another embodiment, the minimum concentration of serum present in the infarct area perfusion-improving composition is at least about 38% expressed as ml/100 cc final volume of the composition. In another embodiment, the minimum concentration of serum present in the infarct area perfusion-improving composition is at least about 39% expressed as ml/100 cc final volume of the composition. In another embodiment, the minimum concentration of serum present in the infarct area perfusion-improving composition is at least about 40% expressed as ml/100 cc final volume of the composition. In another embodiment, the minimum concentration of serum present in the infarct area perfusion-improving composition is at least about 45% expressed as ml/100 cc final volume of the composition. In another embodiment, the minimum concentration of serum present in the infarct area perfusion-improving composition is at least about 50% expressed as ml/100 cc final volume of the composition. In another embodiment, the minimum concentration of serum present in the infarct area perfusion-improving composition is at least about 55% expressed as ml/100 cc final volume of the composition. In another embodiment, the minimum concentration of serum present in the infarct area perfusion-improving composition is at least about 60% expressed as ml/100 cc final volume of the composition. In another embodiment, the minimum concentration of serum present in the infarct area perfusion-improving composition is at least about 65% expressed as ml/100 cc final volume of the composition. In another embodiment, the minimum concentration of serum present in the composition is at least about 70% expressed as ml/100 cc final volume of the composition. In another embodiment, the minimum concentration of serum present in the composition is at least about 75% expressed as ml/100 cc final volume of the composition. In another embodiment, the minimum concentration of serum present in the composition is at least about 80% expressed as ml/100 cc final volume of the composition. In another embodiment, the minimum concentration of serum present in the composition is at least about 85% expressed as ml/100 cc final volume of the composition. In another embodiment, the minimum concentration of serum present in the composition is at least about 90% expressed as ml/100 cc final volume of the composition. In another embodiment, the minimum concentration of serum present in the composition is at least about 95% expressed as ml/100 cc final volume of the composition.
In another embodiment, the maximum concentration of serum present in the infarct area perfusion-improving composition of the described invention is about 100% expressed as ml/100 cc final volume of the composition. In another embodiment, the maximum concentration of serum present in the infarct area perfusion-improving composition of the described invention is about 95% expressed as ml/100 cc final volume of the composition. In another embodiment, the maximum concentration of serum present in the infarct area perfusion-improving composition of the described invention is about 90% expressed as ml/100 cc final volume of the composition. In another embodiment, the maximum concentration of serum present in the infarct area perfusion-improving composition of the described invention is about 85% expressed as ml/100 cc final volume of the composition. In another embodiment, the maximum concentration of serum present in the infarct area perfusion-improving composition of the described invention is about 80% expressed as ml/100 cc final volume of the composition. In another embodiment, the maximum concentration of serum present in the infarct area perfusion-improving composition of the described invention is about 75% expressed as ml/100 cc final volume of the composition. In another embodiment, the maximum concentration of serum present in the infarct area perfusion-improving composition of the described invention is about 70% expressed as ml/100 cc final volume of the composition. In another embodiment, the maximum concentration of serum present in the infarct area perfusion-improving composition of the described invention is about 65% expressed as ml/100 cc final volume of the composition. In another embodiment, the maximum concentration of serum present in the infarct area perfusion-improving composition of the described invention is about 60% expressed as ml/100 cc final volume of the composition. In another embodiment, the maximum concentration of serum present in the infarct area perfusion-improving composition of the described invention is about 55% expressed as ml/100 cc final volume of the composition. In another embodiment, the maximum concentration of serum present in the infarct area perfusion-improving composition of the described invention is about 50% expressed as ml/100 cc final volume of the composition. In another embodiment, the maximum concentration of serum present in the infarct area perfusion-improving composition of the described invention is about 45% expressed as ml/100 cc final volume of the composition. In another embodiment, the maximum concentration of serum present in the infarct area perfusion-improving composition of the described invention is about 40% expressed as ml/100 cc final volume of the composition. In another embodiment, the maximum concentration of serum present in the infarct area perfusion-improving composition of the described invention is about 39% expressed as ml/100 cc final volume of the composition. In another embodiment, the maximum concentration of serum present in the infarct area perfusion-improving composition of the described invention is about 38% expressed as ml/100 cc final volume of the composition. In another embodiment, the maximum concentration of serum present in the infarct area perfusion-improving composition of the described invention is about 37% expressed as ml/100 cc final volume of the composition. In another embodiment, the maximum concentration of serum present in the infarct area perfusion-improving composition of the described invention is about 36% expressed as ml/100 cc final volume of the composition. In another embodiment, the maximum concentration of serum present in the infarct area perfusion-improving composition of the described invention is about 35% expressed as ml/100 cc final volume of the composition. In another embodiment, the maximum concentration of serum present in the infarct area perfusion-improving composition of the described invention is about 34% expressed as ml/100 cc final volume of the composition. In another embodiment, the maximum concentration of serum present in the infarct area perfusion-improving composition of the described invention is about 33% expressed as ml/100 cc final volume of the composition. In another embodiment, the maximum concentration of serum present in the infarct area perfusion-improving composition of the described invention is about 32% expressed as ml/100 cc final volume of the composition. In another embodiment, the maximum concentration of serum present in the infarct area perfusion-improving composition of the described invention is about 31% expressed as ml/100 cc final volume of the composition. In another embodiment, the maximum concentration of serum present in the infarct area perfusion-improving composition of the described invention is about 30% expressed as ml/100 cc final volume of the composition. In another embodiment, the maximum concentration of serum present in the infarct area perfusion-improving composition of the described invention is about 29% expressed as ml/100 cc final volume of the composition. In another embodiment, the maximum concentration of serum present in the infarct area perfusion-improving composition of the described invention is about 28% expressed as ml/100 cc final volume of the composition. In another embodiment, the maximum concentration of serum present in the infarct area perfusion-improving composition of the described invention is about 27% expressed as ml/100 cc final volume of the composition. In another embodiment, the maximum concentration of serum present in the infarct area perfusion-improving composition of the described invention is about 26% expressed as ml/100 cc final volume of the composition. In another embodiment, the maximum concentration of serum present in the infarct area perfusion-improving composition of the described invention is about 25% expressed as ml/100 cc final volume of the composition. In another embodiment, the maximum concentration of serum present in the infarct area perfusion-improving composition of the described invention is about 24% expressed as ml/100 cc final volume of the composition. In another embodiment, the maximum concentration of serum present in the infarct area perfusion-improving composition of the described invention is about 23% expressed as ml/100 cc final volume of the composition. In another embodiment, the maximum concentration of serum present in the infarct area perfusion-improving composition of the described invention is about 22% expressed as ml/100 cc final volume of the composition. In another embodiment, the maximum concentration of serum present in the infarct area perfusion-improving composition of the described invention is about 21% expressed as ml/100 cc final volume of the composition. In another embodiment, the maximum concentration of serum present in the infarct area perfusion-improving composition of the described invention is about 20% expressed as ml/100 cc final volume of the composition. In another embodiment, the maximum concentration of serum present in the infarct area perfusion-improving composition of the described invention is about 15% expressed as ml/100 cc final volume of the composition. In another embodiment, the maximum concentration of serum present in the infarct area perfusion-improving composition of the described invention is about 10% expressed as ml/100 cc final volume of the composition.
In some embodiments, the infarct area perfusion-improving composition may be formulated with an excipient, carrier or vehicle including, but not limited to, a solvent. The terms “excipient”, “carrier”, or “vehicle” as used herein refers to carrier materials suitable for formulation and administration of the chemotactic hematopoietic stem cell product described herein. Carriers and vehicles useful herein include any such materials know in the art which are nontoxic and do not interact with other components. As used herein the phrase “pharmaceutically acceptable carrier” refers to any substantially non-toxic carrier useable for formulation and administration of the composition of the described invention in which the chemotactic hematopoietic stem cell product of the described invention will remain stable and bioavailable.
The pharmaceutically acceptable carrier must be of sufficiently high purity and of sufficiently low toxicity to render it suitable for administration to the mammal being treated. It further should maintain the stability and bioavailability of an active agent. The pharmaceutically acceptable carrier can be liquid or solid and is selected, with the planned manner of administration in mind, to provide for the desired bulk, consistency, etc., when combined with an active agent and other components of a given composition. For example, the pharmaceutically acceptable carrier may be, without limitation, a binding agent (e.g., pregelatinized maize starch, polyvinylpyrrolidone or hydroxypropyl methylcellulose, etc.), a filler (e.g., lactose and other sugars, microcrystalline cellulose, pectin, gelatin, calcium sulfate, ethyl cellulose, polyacrylates, calcium hydrogen phosphate, etc.), a lubricant (e.g., magnesium stearate, talc, silica, colloidal silicon dioxide, stearic acid, metallic stearates, hydrogenated vegetable oils, corn starch, polyethylene glycols, sodium benzoate, sodium acetate, etc.), a disintegrant (e.g., starch, sodium starch glycolate, etc.), or a wetting agent (e.g., sodium lauryl sulfate, etc.). Other suitable pharmaceutically acceptable carriers for the compositions of the described invention include, but are not limited to, water, salt solutions, alcohols, polyethylene glycols, gelatins, amyloses, magnesium stearates, talcs, silicic acids, viscous paraffins, hydroxymethylcelluloses, polyvinylpyrrolidones and the like. Such carrier solutions also can contain buffers, diluents and other suitable additives. The term “buffer” as used herein refers to a solution or liquid whose chemical makeup neutralizes acids or bases without a significant change in pH. Examples of buffers envisioned by the described invention include, but are not limited to, Dulbecco's phosphate buffered saline (PBS), Ringer's solution, 5% dextrose in water (D5W), and normal/physiologic saline (0.9% NaCl). In some embodiments, the infusion solution is isotonic to subject tissues. In some embodiments, the infusion solution is hypertonic to subject tissues. Compositions of the described invention that are for parenteral administration may include pharmaceutically acceptable carriers such as sterile aqueous solutions, non-aqueous solutions in common solvents such as alcohols, or solutions in a liquid oil base.
In some embodiments, the carrier of the infarct area perfusion-improving composition of the described invention may include a release agent such as a sustained release or delayed release carrier. In such embodiments, the carrier may be any material capable of sustained or delayed release of the active to provide a more efficient administration, e.g., resulting in less frequent and/or decreased dosage of the composition, improve ease of handling, and extend or delay effects on diseases, disorders, conditions, syndromes, and the like, being treated, prevented or promoted. Non-limiting examples of such carriers include liposomes, microsponges, microspheres, or microcapsules of natural and synthetic polymers and the like. Liposomes may be formed from a variety of phospholipids such as cholesterol, stearylamines or phosphatidylcholines.
The infarct area perfusion-improving compositions of the described invention may be administered parenterally in the form of a sterile injectable aqueous or oleaginous suspension. The term “parenteral” or “parenterally” as used herein refers to introduction into the body by way of an injection (i.e., administration by injection), including, but not limited to, infusion techniques. The infarct area perfusion-improving composition of the described invention comprising a chemotactic hematopoietic stem cell product is delivered to the subject by means of a balloon catheter adapted for delivery of the fluid compositions (i.e., compositions capable of flow) into a selected anatomical structure.
The sterile infarct area perfusion-improving composition of the described invention may be a sterile solution or suspension in a nontoxic parenterally acceptable diluent or solvent. A solution generally is considered as a homogeneous mixture of two or more substances; it is frequently, though not necessarily, a liquid. In a solution, the molecules of the solute (or dissolved substance) are uniformly distributed among those of the solvent. A suspension is a dispersion (mixture) in which a finely-divided species is combined with another species, with the former being so finely divided and mixed that it does not rapidly settle out. In everyday life, the most common suspensions are those of solids in liquid water. Among the acceptable vehicles and solvents that may be employed are water, Ringer's solution, and isotonic sodium chloride (saline) solution. In some embodiments, hypertonic solutions are employed. In addition, sterile, fixed oils conventionally are employed as a solvent or suspending medium. For parenteral application, particularly suitable vehicles consist of solutions, preferably oily or aqueous solutions, as well as suspensions, emulsions, or implants. Aqueous suspensions may contain substances, which increase the viscosity of the suspension and include, for example, sodium carboxymethyl cellulose, sorbitol and/or dextran.
Additional infarct area perfusion-improving compositions of the described invention readily may be prepared using technology, which is known in the art, such as described in Remington's Pharmaceutical Sciences, 18th or 19th editions, published by the Mack Publishing Company of Easton, Pa., which is incorporated herein by reference.
As used herein the terms “therapeutically effective”, “infarct area-improving amount”, “infarct area-improving amount”, “perfusion improving amount” or “pharmaceutically effective amount” refer to the amount of the compositions of the invention that result in a therapeutic or beneficial effect following its administration to a subject. The infarct area-improving, infarct area-improving, perfusion-improving, therapeutic, or pharmaceutical effect may be curing, minimizing, preventing or ameliorating a disease or disorder, or may have any other infarct area-improving, infarct area-improving, perfusion-improving, therapeutic, or pharmaceutical beneficial effect. The concentration of the substance is selected so as to exert its infarct area-improving, infarct area-improving, perfusion-improving, therapeutic, or pharmaceutical effect, but low enough to avoid significant side effects within the scope and sound judgment of the physician. The effective amount of the composition may vary with the age and physical condition of the biological subject being treated, the severity of the condition, the duration of the treatment, the nature of concurrent therapy, the timing of the infusion, the specific compound, composition or other active ingredient employed, the particular carrier utilized, and like factors.
A skilled artisan may determine a pharmaceutically effective amount of the inventive compositions by determining the dose in a dosage unit (meaning unit of use) that elicits a given intensity of effect, hereinafter referred to as the “unit dose.” The term “dose-intensity relationship” refers to the manner in which the intensity of effect in an individual recipient relates to dose. The intensity of effect generally designated is 50% of maximum intensity. The corresponding dose is called the 50% effective dose or individual ED50. The use of the term “individual” distinguishes the ED50 based on the intensity of effect as used herein from the median effective dose, also abbreviated ED50, determined from frequency of response data in a population. “Efficacy” as used herein refers to the property of the compositions of the described invention to achieve the desired response, and “maximum efficacy” refers to the maximum achievable effect. The amount of the chemotactic hematopoietic stem cell product in the pharmaceutical compositions of the described invention that will be effective in the treatment of a particular disorder or condition will depend on the nature of the disorder or condition, and may be determined by standard clinical techniques. (See, for example, Goodman and Gilman's THE PHARMACOLOGICAL BASIS OF THERAPEUTICS, Joel G. Hannan, Lee E. Limbird, Eds.; McGraw Hill, New York, 2001; THE PHYSICIAN'S DESK REFERENCE, Medical Economics Company, Inc., Oradell, N.J., 1995; and DRUG FACTS AND COMPARISONS, FACTS AND COMPARISONS, INC., St. Louis, Mo., 1993). The precise dose to be employed in the formulations of the described invention also will depend on the route of administration and the seriousness of the disease or disorder, and should be decided according to the judgment of the practitioner and each subject's circumstances. It is envisioned that subjects may benefit from multiple administrations of the pharmaceutical composition of the described invention.
In another embodiment, the pharmaceutical compositions according to the described invention contain at least 10×106 CD34+ hematopoietic stem cells having a subpopulation of at least 0.5×106 CD34+ cells expressing CXCR-4 and having CXCR-4 mediated chemotactic activity per dosage unit for parenteral administration at the physician's discretion.
In another aspect of the described invention, the infarct area perfusion-improving pharmaceutical compositions of the described invention may further include one or more compatible active ingredients, which are aimed at providing the infarct area perfusion-improving composition with another pharmaceutical effect in addition to that provided by the isolated chemotactic hematopoietic stem cell product of the described invention. “Compatible” as used herein means that the active ingredients of such a composition are capable of being combined with each other in such a manner so that there is no interaction that would substantially reduce the efficacy of each active ingredient or the composition under ordinary use conditions. In some embodiments, the combination therapy comprises administering to a subject in need thereof a infarct area perfusion-improving pharmaceutical composition comprising a chemotactic hematopoietic stem cell product of the described invention combined with an agent selected from the group consisting of an angiotensin converting enzyme (ACE) inhibitor, a beta-blocker, a diuretic, an anti-arrhythmic agent, an anti-anginal agent, a tyrosine kinase receptor agonist, a vasoactive agent or inotrope, an anticoagulant agent, a fibrinolytic agent, and a hypercholesterolemic agent.
In some embodiments, the tyrosine kinase receptor agonist is neuregulin 1. Neuregulin 1 (NRG1) is an agonist for receptor tyrosine kinases of the epidermal growth factor receptor family, consisting of ErbB1, 2, 3, and 4. (Fuller, S J, et al., J. Mol. Cell Cariol. 44: 831-54 (2008). Binding of NRG1 to Erb4 increases its kinase activity and leads to heterodimerization with erbB2 or homodimerization with ErbB4 and stimulation of intracellular signal transduction pathways. Id. NFRG1 receptor subunits ErbB2 and ErbB4 also are expressed in differentiated cardiomyocytes. Id. Recently it has been shown in mice that NRG1 induces proliferation of differentiated mononucleated cardiomyocytes in vivo by inducing differentiated cardiomyocytes to leave proliferative quiescence. Bersell, et al (Bersell, K. et al., Cell 138: 257-70 (2009). Undifferentiated stem and progenitor cells did not contribute to this proliferation. (Id). Using a mouse model in which the left anterior descending coronary artery (LAD) of two month old mice was ligated permanently and NRG1 administered daily one week later for 12 weeks, it was shown that administration of NRG1 for 12 weeks resulted in a sustained improvement in myocardial function, determined by ejection fraction, a reduced infarct scar size, and attenuation of cardiomyocyte hypertrophy. (Id).
Following acute myocardial infarction, in addition to necrotic cell death as a consequence of ischemia, ongoing apoptotic cell death and cardiomyocyte hibernation collectively lead to a decrement in cardiac function that can worsen over time and ultimately causing major adverse cardiac events. Once lost, cardiomyocytes are unable to significantly regenerate to restore cardiac function. Carbon 14 dating of cardiomyocytes show the regenerative capacity of cardiac muscle to be less than 1% annually (Bergman O, Science. 2009; 324:98-101).
In some embodiments, the composition of the described invention further comprises about 0.5% to about 5% albumin. In some embodiments, the minimum amount of albumin is about 0.5% expressed as ml/100 cc volume of the composition. In some embodiments, the minimum amount of albumin is about 0.75% expressed as ml/100 cc volume of the composition. In some embodiments, the minimum amount of albumin is about 1.0% expressed as ml/100 cc volume of the composition. In some embodiments, the minimum amount of albumin is about 1.25% expressed as ml/100 cc volume of the composition. In some embodiments, the minimum amount of albumin is about 1.5% expressed as ml/100 cc volume of the composition. In some embodiments, the minimum amount of albumin is about 1.75% expressed as ml/100 cc volume of the composition. In some embodiments, the minimum amount of albumin is about 2.0% expressed as ml/100 cc volume of the composition. In some embodiments, the minimum amount of albumin is about 2.5% expressed as ml/100 cc volume of the composition. In some embodiments, the minimum amount of albumin is about 2.75% expressed as ml/100 cc volume of the composition. In some embodiments, the minimum amount of albumin is about 3.0% expressed as ml/100 cc volume of the composition. In some embodiments, the minimum amount of albumin is about 3.5% expressed as ml/100 cc volume of the composition. In some embodiments, the minimum amount of albumin is about 4.0% expressed as ml/100 cc volume of the composition. In some embodiments, the minimum amount of albumin is about 4.5% expressed as ml/100 cc volume of the composition. In some embodiments, the minimum amount of albumin is about 5.0% expressed as ml/100 cc volume of the composition.
In some embodiments, the maximum amount of albumin in the compositions of the described invention is about 5.0% expressed as ml/100 cc volume of the composition. In some embodiments, the maximum amount of albumin in the compositions of the described invention is about 4.75% expressed as ml/100 cc volume of the composition. In some embodiments, the maximum amount of albumin in the compositions of the described invention is about 4.5% expressed as ml/100 cc volume of the composition. In some embodiments, the maximum amount of albumin in the compositions of the described invention is about 4.0% expressed as ml/100 cc volume of the composition. In some embodiments, the maximum amount of albumin in the compositions of the described invention is about 4.25% expressed as ml/100 cc volume of the composition. In some embodiments, the maximum amount of albumin in the compositions of the described invention is about 4.0% expressed as ml/100 cc volume of the composition. In some embodiments, the maximum amount of albumin in the compositions of the described invention is about 3.75% expressed as ml/100 cc volume of the composition. In some embodiments, the maximum amount of albumin in the compositions of the described invention is about 3.5% expressed as ml/100 cc volume of the composition. In some embodiments, the maximum amount of albumin in the compositions of the described invention is about 3.25% expressed as ml/100 cc volume of the composition. In some embodiments, the maximum amount of albumin in the compositions of the described invention is about 3.0% expressed as ml/100 cc volume of the composition. In some embodiments, the maximum amount of albumin in the compositions of the described invention is about 2.75% expressed as ml/100 cc volume of the composition. In some embodiments, the maximum amount of albumin in the compositions of the described invention is about 2.0% expressed as ml/100 cc volume of the composition. In some embodiments, the maximum amount of albumin in the compositions of the described invention is about 1.75% expressed as ml/100 cc volume of the composition. In some embodiments, the maximum amount of albumin in the compositions of the described invention is about 1.5% expressed as ml/100 cc volume of the composition. In some embodiments, the maximum amount of albumin in the compositions of the described invention is about 1.25% expressed as ml/100 cc volume of the composition. In some embodiments, the maximum amount of albumin in the compositions of the described invention is about 1% expressed as ml/100 cc volume of the composition. In some embodiments, the albumin is human albumin. In some embodiments the albumin is recombinant human albumin.
Methods of the Described Invention
In another aspect, the described invention provides a method of preparing a infarct area perfusion-improving pharmaceutical composition comprising a chemotactic hematopoietic stem cell product for treating a subject in need thereof. The method comprises the steps of
(1) acquiring a preparation comprising an enriched population of potent CD34+ cells from the subject under sterile conditions by a chemotactic cell acquisition process;
(2) sterilely purifying potent CD34+ cells containing a subpopulation of potent cells expressing CXCR-4 and having CXCR-4-mediated chemotactic activity from the preparation;
(3) sterilely formulating the purified potent CD34+ cells to form the chemotactic hematopoietic stem cell product;
(4) sterilely formulating the chemotactic hematopoietic stem cell product containing a subpopulation of potent CD34+CXCR-4+ cells having chemotactic activity to form a pharmaceutical composition;
(5) assessing sterility of the pharmaceutical composition;
(6) releasing the sterile pharmaceutical composition as eligible for infusion into the subject;
(7) loading a therapeutically effective amount of the pharmaceutical composition into a delivery apparatus; and
(8) optionally transporting the delivery apparatus containing the therapeutically effective amount of the sterile pharmaceutical composition comprising the chemotactic hematopoietic stem cell product to a cardiac catheterization facility for infusion in to the subject.
In one embodiment, step (2) is initiated within about 12 hours to about 24 hours of completion of acquiring step (1). In another embodiment, releasing step (7) proceeds only if the sterile formulated cell product is to be infused into the subject within about 48 hours to about 72 hours of completion of acquiring step (1). In another embodiment, step (2) is initiated within about 12 hours to about 24 hours of completion of acquiring step (1), and releasing step (6) proceeds only if the sterile formulated cell product is to be infused into the subject within about 48 hours to about 72 hours of completion of acquiring step (1).
In one embodiment, step (5), i.e., the step of assessing sterility of the pharmaceutical composition, further comprises the steps of (i) centrifuging the chemotactic hematopoietic stem cell product comprising potent CD34+CXCR-4+ cells to form a cell pellet and a supernatant, the cell pellet comprising the potent CD34+CXCR-4+ cells; (ii) sterilely removing the supernatant without disturbing the cell pellet; and (iii) analyzing whether the supernatant is contaminated by a microbe thereby determining the sterility of the cell pellet without exhausting its cell content.
In one embodiment, in step (a), the chemotactic cell acquisition process is a mini-bone marrow harvest technique used to acquire a preparation comprising an enriched population of potent CD34+ cells from the bone marrow of the subject under sterile conditions. For the bone marrow harvest technique, step (a) of the method further comprises the steps: (i) preloading harvesting syringes with heparin prior to harvesting bone marrow from a subject; (ii) aspirating the bone marrow from a left posterior iliac crest and a right posterior iliac crest of the subject using the harvesting syringes and a mini-bone marrow harvest technique to form harvested bone marrow; and (iii) infusing the harvested bone marrow into a collecting bag. In one embodiment, the harvesting syringes in step (i) and the collecting bag in step (iii) contain a preservative free heparinized solution comprising 0.9% normal saline. The final concentration of heparin in the heparinized saline solution is about 20 units per ml to about 25 units per ml.
Optionally, in one embodiment of the method, the harvested bone marrow is transported to a processing facility different from the facility from which the bone marrow was harvested. In one embodiment, the method for transporting the harvested bone marrow to the processing facility comprises the steps (a) placing the harvested bone marrow in a collection bag; (b) placing the collection bag in a secondary bag; (c) placing the secondary bag containing the collection bag in a shipping container comprising an interior compartment containing frozen wet ice and at least one sheet of bubble wrap; (d) affixing a temperature tag monitor to the interior compartment of the shipping container; (e) sealing the shipping container; and (f) shipping the shipping container to the processing facility.
In another aspect, the described invention provides a method of treating or repairing an infarct area injury in a revascularized subject following an acute myocardial infarction resulting from a natural disease process the method comprising the steps: (a) administering to the subject parenterally through a catheter a sterile pharmaceutical composition comprising: (i) an infarct area perfusion-improving amount of a nonexpanded sterile isolated chemotactic hematopoietic stem cell product as a first therapeutic agent, wherein the infarct area perfusion-improving amount of the chemotactic hematopoietic stem cell product comprises an enriched population of at least 10×106 isolated autologous CD34+ hematopoietic stem cells containing a subpopulation of at least 0.5×106 potent CD34+ cells expressing CXCR-4 and having CXCR-4 mediated chemotactic activity; (ii) a stabilizing amount of serum, wherein the stabilizing amount of serum is greater than 20% (v/v), and (iii) optionally a therapeutically effective amount of at least one compatible second therapeutic agent; wherein at least 70% of cells in the enriched population of isolated CD34+ cells containing the subpopulation of potent cells that express CXCR-4 and that have CXCR-4-mediated chemotactic activity when passed through the catheter and when tested in vitro are CD34+ cells, and wherein the enriched population of isolated CD34+ cells containing a subpopulation of potent cells that express CXCR-4 and that have CXCR-4-mediated chemotactic activity when passed through the catheter and tested in vitro (1) retains the CXCR-4-mediated chemotactic activity; (2) is at least about 70% viable; and (3) is able to form hematopoietic colonies in vitro, for at least about 24 hours following acquisition from the subject of the enriched population of CD34+ cells containing the subpopulation of potent cells that express CXCR-4; and (b) improving perfusion in an infarct area, relative to controls, wherein administering step (a) occurs at one or more infusion dates and the first infusion date comprises a specific time interval defined by a first time and a second time, wherein the first time is after peak inflammatory cytokine cascade production in the infarcted area and the second time is before myocardial scar formation in the infarcted area.
According to one embodiment, the therapeutically effective amount of the chemotactic hematopoietic stem cell product comprises at least 15×106 isolated CD34+ hematopoietic stem cells containing a subpopulation of at least 0.5×106 potent CD34+ cells expressing CXCR-4 and having CXCR-4 mediated chemotactic activity. According to another embodiment, the enriched population of CD34+ cells containing a subpopulation of potent cells expressing CXCR-4 and having CXCR-4 mediated chemotactic activity (a) is capable of forming hematopoietic colonies in vitro; and (b) retains at least 2% of the CXCR-4-mediated chemotactic activity, for at least 48 hours following acquisition of the of the enriched population of CD34+ cells containing a subpopulation of potent cells that express CXCR-4 in (a). According to another embodiment, the enriched population of CD34+ cells containing a subpopulation of potent cells expressing CXCR-4 and having CXCR-4 mediated chemotactic activity (a) is capable of forming hematopoietic colonies in vitro; and (b) retains at least 2% of the CXCR-4-mediated chemotactic activity, for at least 72 hours following acquisition of the enriched population of CD34+ cells containing a subpopulation of potent cells that express CXCR-4 in (a). According to another embodiment, the subpopulation of potent cells expressing CXCR-4 and having CXCR-4 mediated chemotactic activity retains at least 2% of the CXCR-4-mediated chemotactic activity for at least 24 hours following acquisition from the subject of the enriched population of CD34+ cells containing a subpopulation of potent cells that express CXCR-4 in (a).
According to some embodiments, the optional second therapeutic agent is selected from the group consisting of an angiotensin converting enzyme inhibitor, a beta-blocker, a diuretic, an anti-arrhythmic agent, an anti-anginal agent, a tyrosine kinase receptor agonist, a vasoactive agent, an anticoagulant agent, a fibrinolytic agent, and a hypercholesterolemic agent. According to some embodiments, the tyrosine kinase receptor agonist is neuregulin 1. According to some embodiments, the infarct area injury is a progressive decline in heart muscle function following the acute myocardial infarction.
According to another embodiment, the method reduces at least one infarct area injury more than the composition components (i) plus (ii) or component (iii) alone. According to some embodiments, the infarct area injury comprises apoptotic cardiomyocyte loss in the infarct area. According to some embodiments, the infarct area injury comprises adverse ventricular remodeling after an acute myocardial infarction. According to some embodiments, the infarct area injury comprises a progressive decline in heart muscle function resulting from the acute myocardial infarction. According to some embodiments, the method increases perfusion of at least one ischemic peri-infarct zone of myocardial tissue, when compared to controls. According to some embodiments, the method increases perfusion to hibernating myocardium in at least one peri-infarct zone of myocardial tissue, when compared to controls. According to some embodiments, the infarct area injury comprises hypoperfusion in the peri-infarct border zone, relative to controls. According to some embodiments, the infarct area injury comprises myocardial hibernation in the peri-infarct border zone, relative to controls. According to some embodiments, the method improves infarct area microvascular blood flow, when compared to controls. According to some embodiments, the method decreases the infarct area, when compared to controls. According to some embodiments, the method decreases infarct mass, when compared to controls.
According to one embodiment of the described invention, the subject in need thereof is a revascularized myocardial infarction patient. The term “revascularized” as used in this embodiment refers to the successful placement of a stent. Clinical evaluations, for example, of coronary insufficiency using non-laboratory tests, cardiac catheterization, measurement of inflammatory cytokines, and measurement of cardiac biomarkers may be used to determine the appropriate time to administer the pharmaceutical compositions in accordance with the methods of the described invention. In some embodiments, detection of peak inflammatory cytokine cascade production enables the administration to be tailored at the window most crucial for the particular subject. In some embodiments, peak inflammatory cytokine cascade production is determined by measuring the levels of the appropriate cytokine(s) in the plasma and or urine. In other embodiments, the level(s) of the appropriate cytokine(s) is/are measured immunochemically, for example, by a sandwich enzyme immunoassay, by enzyme-linked immunosorbent assays (ELISA) or by multiplex bead kits.
According to one embodiment, the composition is administered to the subject at a first infusion date after an inflammatory cytokine cascade production peaks. In some embodiments, the composition is administered to the revascularized myocardial infarction patient at a first infusion date about 5 days to about 14 days post-infarction. The minimum time in which to administer the composition to the revascularized myocardial infarction patient at the first infusion date is about 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 days. The maximum time in which to administer the composition at the first infusion date is about 14, 12, 11, 10, 9, 8, 7, 6, or 5 days.
According to some embodiments, the minimum number of CD34+ cells in the enriched population of CD34+ cells containing a subpopulation of potent CD34+ cells expressing CXCR-4 and having CXCR-4 mediated chemotactic activity is that number of cells that provides a sufficient number of potent CD34+ cells expressing CXCR-4 and having CXCR-4-mediated motility to produce the infarct-area perfusion-improving effect. Thus, the described invention contemplates that in some embodiments, where expression of CXCR-4 and CXCR-4 mediated mobility may be increased, for example, by selection and enrichment of CXCR-4+ motile cells, a lesser number of CD34+ cells may be necessary to produce the infarct-area perfusion-improving effect. According to some such embodiments, an enriched population of at least 1×105 isolated autologous CD34+ hematopoietic stem cells provides a sufficient number of potent CD34+ cells expressing CXCR-4 and having CXCR-4-mediated motility to produce the infarct-area perfusion-improving effect. According to some such embodiments, at least 5×105 isolated autologous CD34+ hematopoietic stem cells provides a sufficient number of potent CD34+ cells expressing CXCR-4 and having CXCR-4-mediated motility to produce the infarct-area perfusion-improving effect. According to some such embodiments, at least 9×105 isolated autologous CD34+ hematopoietic stem cells provides a sufficient number of potent CD34+ cells expressing CXCR-4 and having CXCR-4-mediated motility to produce the infarct-area perfusion-improving effect. According to some such embodiments, at least 1×106 isolated autologous CD34+ hematopoietic stem cells provides a sufficient number of potent CD34+ cells expressing CXCR-4 and having CXCR-4-mediated motility to produce the infarct-area perfusion-improving effect. According to some such embodiments, at least 2×106 isolated autologous CD34+ hematopoietic stem cells provides a sufficient number of potent CD34+ cells expressing CXCR-4 and having CXCR-4-mediated motility to produce the infarct-area perfusion-improving effect. According to some such embodiments, at least 3×106 isolated autologous CD34+ hematopoietic stem cells provides a sufficient number of potent CD34+ cells expressing CXCR-4 and having CXCR-4-mediated motility to produce the infarct-area perfusion-improving effect. According to some such embodiments, at least 4×106 isolated autologous CD34+ hematopoietic stem cells provides a sufficient number of potent CD34+ cells expressing CXCR-4 and having CXCR-4-mediated motility to produce the infarct-area perfusion-improving effect. According to some such embodiments, at least 5×106 isolated autologous CD34+ hematopoietic stem cells provides a sufficient number of potent CD34+ cells expressing CXCR-4 and having CXCR-4-mediated motility to produce the infarct-area perfusion-improving effect. According to some such embodiments, at least 6×106 isolated autologous CD34+ hematopoietic stem cells provides a sufficient number of potent CD34+ cells expressing CXCR-4 and having CXCR-4-mediated motility to produce the infarct-area perfusion-improving effect. According to some such embodiments, at least 7×106 isolated autologous CD34+ hematopoietic stem cells provides a sufficient number of potent CD34+ cells expressing CXCR-4 and having CXCR-4-mediated motility to produce the infarct-area perfusion-improving effect. According to some such embodiments, at least 8×106 isolated autologous CD34+ hematopoietic stem cells provides a sufficient number of potent CD34+ cells expressing CXCR-4 and having CXCR-4-mediated motility to produce the infarct-area perfusion-improving effect. According to some such embodiments, at least 9×106 isolated autologous CD34+ hematopoietic stem cells provides a sufficient number of potent CD34+ cells expressing CXCR-4 and having CXCR-4-mediated motility to produce the infarct-area perfusion-improving effect.
According to some embodiments, an infarct area perfusion-improving amount of CD34+ cells containing a subpopulation of at least 0.5×106 potent CD34+ cells expressing CXCR-4 and having CXCR-4 mediated chemotactic activity comprises an enriched population of at least 10×106 isolated autologous CD34+ hematopoietic stem cells containing a subpopulation of at least 0.5×106 potent CD34+ cells expressing CXCR-4 and having CXCR-4 mediated chemotactic activity is infused at a second infusion date. According to some such embodiments, the second infusion date is at least 5 days after the first infusion date. According to some such embodiments, the second infusion date is at least 6 days after the first infusion date. According to some such embodiments, the second infusion date is at least 7 days after the first infusion date. According to some such embodiments, the second infusion date is at least 8 days after the first infusion date. According to some such embodiments, the second infusion date is at least 9 days after the first infusion date. According to some such embodiments, the second infusion date is at least 10 days after the first infusion date. According to some such embodiments, the second infusion date is at least 11 days after the first infusion date. According to some such embodiments, the second infusion date is at least 12 days after the first infusion date. According to some such embodiments, the second infusion date is at least 13 days after the first infusion date. According to some such embodiments, the second infusion date is at least 14 days after the first infusion date. According to some such embodiments, the second infusion date is at least 15 days after the first infusion date. According to some such embodiments, the second infusion date is at least 15 days after the first infusion date. According to some such embodiments, the second infusion date is at least 16 days after the first infusion date. According to some such embodiments, the second infusion date is at least 17 days after the first infusion date. According to some such embodiments, the second infusion date is at least 18 days after the first infusion date. According to some such embodiments, the second infusion date is at least 19 days after the first infusion date. According to some such embodiments, the second infusion date is at least 20 days after the first infusion date. According to some such embodiments, the second infusion date is at least 21 days after the first infusion date. According to some such embodiments, the second infusion date is at least 22 days after the first infusion date. According to some such embodiments, the second infusion date is at least 23 days after the first infusion date. According to some such embodiments, the second infusion date is at least 24 days after the first infusion date. According to some such embodiments, the second infusion date is at least 25 days after the first infusion date. According to some such embodiments, the second infusion date is at least 26 days after the first infusion date. According to some such embodiments, the second infusion date is at least 27 days after the first infusion date. According to some such embodiments, the second infusion date is at least 28 days after the first infusion date. According to some such embodiments, the second infusion date is at least 29 days after the first infusion date. According to some such embodiments, the second infusion date is at least 30 days after the first infusion date. According to some such embodiments, the second infusion date is at least 31 days after the first infusion date. According to some such embodiments, the second infusion date is at least 32 days after the first infusion date. According to some such embodiments, the second infusion date is at least 33 days after the first infusion date. According to some such embodiments, the second infusion date is at least 34 days after the first infusion date. According to some such embodiments, the second infusion date is at least 35 days after the first infusion date. According to some such embodiments, the second infusion date is at least 36 days after the first infusion date. According to some such embodiments, the second infusion date is at least 37 days after the first infusion date. According to some such embodiments, the second infusion date is at least 38 days after the first infusion date. According to some such embodiments, the second infusion date is at least 39 days after the first infusion date. According to some such embodiments, the second infusion date is at least 40 days after the first infusion date. According to some such embodiments, the second infusion date is at least 45 days after the first infusion date. According to some such embodiments, the second infusion date is at least 50 days after the first infusion date. According to some such embodiments, the second infusion date is at least 55 days after the first infusion date. According to some such embodiments, the second infusion date is at least 60 days after the first infusion date.
According to some embodiments, the infarct area perfusion-improving amount of the chemotactic hematopoietic stem cell product comprising an enriched population of at least 10×106 isolated autologous CD34+ hematopoietic stem cells containing a subpopulation of at least 0.5×106 potent CD34+ cells expressing CXCR-4 and having CXCR-4 mediated chemotactic activity is infused at a third infusion date. According to some such embodiments, the third infusion date is at least 30 days after the first infusion date. According to some such embodiments, the third infusion date is at least 31 days after the first infusion date. According to some such embodiments, the third infusion date is at least 32 days after the first infusion date. According to some such embodiments, the third infusion date is at least 33 days after the first infusion date. According to some such embodiments, the third infusion date is at least 34 days after the first infusion date. According to some such embodiments, the third infusion date is at least 35 days after the first infusion date. According to some such embodiments, the third infusion date is at least 36 days after the first infusion date. According to some such embodiments, the third infusion date is at least 37 days after the first infusion date. According to some such embodiments, the third infusion date is at least 38 days after the first infusion date. According to some such embodiments, the third infusion date is at least 39 days after the first infusion date. According to some such embodiments, the third infusion date is at least 40 days after the first infusion date. According to some such embodiments, the third infusion date is at least 45 days after the first infusion date. According to some such embodiments, the third infusion date is at least 50 days after the first infusion date. According to some such embodiments, the third infusion date is at least 55 days after the first infusion date. According to some such embodiments, the third infusion date is at least 60 days after the first infusion date. According to some such embodiments, the third infusion date is at least 61 days after the first infusion date. According to some such embodiments, the third infusion date is at least 62 days after the first infusion date. According to some such embodiments, the third infusion date is at least 63 days after the first infusion date. According to some such embodiments, the third infusion date is at least 64 days after the first infusion date. According to some such embodiments, the third infusion date is at least 65 days after the first infusion date. According to some such embodiments, the third infusion date is at least 66 days after the first infusion date. According to some such embodiments, the third infusion date is at least 67 days after the first infusion date. According to some such embodiments, the third infusion date is at least 68 days after the first infusion date. According to some such embodiments, the third infusion date is at least 69 days after the first infusion date. According to some such embodiments, the third infusion date is at least 70 days after the first infusion date. According to some such embodiments, the third infusion date is at least 75 days after the first infusion date. According to some such embodiments, the third infusion date is at least 75 days after the first infusion date. According to some such embodiments, the third infusion date is at least 80 days after the first infusion date. According to some such embodiments, the third infusion date is at least 85 days after the first infusion date. According to some such embodiments, the third infusion date is at least 90 days after the first infusion date.
In some embodiments, the delivery apparatus used to deliver the pharmaceutical composition of the described invention to a subject in need thereof comprises an infusion syringe, a flushing syringe, a four-way stopcock, and a balloon catheter. In one embodiment, the delivery apparatus comprises (a) an infusion device attached to a sterile four-way stopcock containing the pharmaceutical composition comprising the chemotactic hematopoietic stem cell product; (b) a flushing device attached to the sterile four-way stopcock, the flushing device containing a flushing solution, and (c) a catheter attached to the delivery apparatus by the sterile four-way stopcock. According to one embodiment, the infusion device is a syringe made of any suitable material. The body and handle of suitable four way stopcocks may be made of the same or a different material. Examples of suitable four-way stopcocks includes, without limitation, a stopcock having a polycarbonate body/polycarbonate handle, a stopcock having a polyethylene body/polyethylene handle, a stopcock having a polycarbonate body/polyethylene handle, or a disposable stopcock. In another embodiment, a device is further attached to the stopcock to regulate the pressure exerted on the delivered solution. In some embodiments an integral flush device or syringe is attached to the stopcock. In one embodiment, the catheter is a balloon catheter. The term “balloon catheter” refers to a type of “soft” thin flexible tube having an inflatable “balloon” at its tip, which is used during a catheterization procedure to enlarge a narrow opening or passage within the body. The deflated balloon catheter is positioned, inflated to perform the necessary procedure, and deflated again to be removed.
The viability and potential efficacy of the chemotactic hematopoietic stem cell product of the described invention comprising potent CD34+CXCR-4+ cells depends on the cells maintaining their potency as they pass through a catheter. The catheter used in the methods of the described invention has an internal diameter of at least 0.36 mm. Any type of catheter having an internal diameter of at least 0.36 mm may be effective in delivering the pharmaceutical compositions of the described invention.
For example, a flow control catheter, which slows drainage of blood through the coronary artery vasculature, allows the cells time to transit through the blood vessel wall and into the tissue.
In some embodiments, the catheter is a balloon catheter. For example, without limitation, the following balloon dilatation catheters available from Cordis, Boston Scientific, Medtronic and Guidant having an internal diameter of about 0.36 mm have been validated (see Table 1).







TABLE 1







Balloon catheter validated for infusion of


selected CD34+ cells through the IRA










Manu-
Name and Model
Balloon
Lumen Internal


facturer
No.
Dimensions
Diameter





Cordis
Raptor OTW
15 mm × 3.0 mm
0.36 mm (0.14 in.)



579-130


Boston
OTW Maverick
15 mm × 3.0 mm
0.36 mm (0.14 in.)


Scientific
20620-1530


Medtronic
OTW Sprinter SPR
15 mm × 3.0 mm
0.36 mm (0.14 in.)



3015W


Guidant
Voyager OTW
15 mm × 3.0 mm
0.36 mm (0.14 in.)



1009443-15









In addition, catheters have been described having a fluid delivery port adjacent to the balloon such that the balloon may be inflated against a vessel wall to isolate the delivery site from hemodynamics opposite the balloon from the port, which may be located distally of the balloon. Additionally, balloon catheters have been disclosed having lumens ending in side ports disposed proximally to the balloon catheter; these balloon catheters generally may be referred to as “balloon/delivery” catheters, although particular references may use different descriptors. See, e.g., U.S. Pat. No. 5,415,636 to Forman, incorporated by reference herein.
In some embodiments, the method of treating or repairing an infarct area injury in the aftermath of an acute myocardial infarction resulting from a natural disease process comprises administering the infarct area perfusion-improving pharmaceutical composition via balloon catheterization intravascularly (meaning inside a blood vessel) into an infarcted artery. In some embodiments, following angioplasty, a delivery balloon catheter is inserted via a femoral artery into a desired coronary artery, such as the left anterior descending coronary artery. Some medical conditions may require both a balloon catheter and a fluid delivery catheter to facilitate treatment. In some embodiments, a catheter is used to directly inject cells into the myocardium.
Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range is encompassed within the invention. The upper and lower limits of these smaller ranges which may independently be included in the smaller ranges also is encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either both of those included limits are also included in the invention.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the described invention, the preferred methods and materials are now described. All publications mentioned herein are incorporated herein by reference to disclose and describe the methods and/or materials in connection with which the publications are cited.
As used herein and in the appended claims, the singular forms “a”, “and”, and “the” include plural referents unless the context clearly dictates otherwise. All technical and scientific terms used herein have the same meaning.
Each of the references cited herein is incorporated herein by reference in its entirety. The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the described invention is not entitled to antedate such publication by virtue of prior invention. Further, the dates of publication provided may be different from the actual publication dates which may need to be confirmed independently.
EXAMPLES
The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the described invention, and are not intended to limit the scope of what the inventors regard as their invention nor are they intended to represent that the experiments below are all or the only experiments performed. Efforts have been made to ensure accuracy with respect to numbers used (e.g. amounts, temperature, etc.) but some experimental errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, molecular weight is weight average molecular weight, temperature is in degrees Centigrade, and pressure is at or near atmospheric.
Phase I Clinical Trial Protocol
Example 1
Selection of Eligible Subjects
Subjects/patients presenting with symptoms and clinical findings suggestive of a myocardial infarction received emergency diagnostic and clinical management according to institutional guidelines. If a transmural (meaning through the wall) myocardial infarction was confirmed, the time of first symptoms and the time of successful stent placement was recorded. Revascularized subjects received appropriate medical management to reduce ventricular wall stresses according to institutional guidelines. The term “revascularized” as used in this embodiment, refers to the successful placement of a stent.
All types of stents, including drug-eluting stents (e.g., paclitaxel or sirolimus) are acceptable for use in the revascularization of the infarct related artery (“IRA”). Previous studies employing balloon catheters to infuse cell products have reported no limits for reference vessel diameter for the placement of the stent. Since this study was designed to distribute the cell product into the IRA circulation, and in an attempt to limit the potential for damage to very small vessels, the described invention required that stents be placed prior to infusion of the chemotactic hematopoietic stem cell product of the described invention.
Stent-related drug effects occur predominantly at the site of contact of the stent with the vessel wall. Consequent to balloon dilatation, there is limited blood flow across the stent during cell infusion, and therefore no significant adverse drug-mediated effect on the CD34+ cells in the chemotactic hematopoietic stem cell product was expected. Moreover, prior clinical studies have shown that by 96 hours after drug-eluting stent placement, whole blood levels of either paclitaxel or sirolimus are below the limits of detection. Therefore, tissue levels in the myocardial sites to which the infused CD34+ cells expressing CXCR-4 and having CXCR-4-mediated chemotactic activity were intended to migrate were expected to be inconsequential. See Sousa, J. et al., Circulation 107: 2274-79, 2383-89 (2003).
During revascularization, a subject's cardiac function and perfusion was assessed by standard methods. Relevant measures of cardiac function following a myocardial infarction include assessment of global ejection fraction, ventricular volumes, resting perfusion, and infarct size.
The term “ejection fraction” (“EF”) refers to the percentage of blood emptied from the ventricle during contraction More specifically, it is the fraction of the end-diastolic volume that is ejected with each beat; that is, it is stroke volume (SV) divided by end-diastolic volume (EDV). The volume of blood within a ventricle immediately before a contraction is known as the end-diastolic volume, while the volume of blood left in a ventricle at the end of contraction is known as end-systolic volume. The difference between end-diastolic and end-systolic volumes is the stroke volume, the volume of blood ejected with each beat. In a healthy 70-kg (154-1b) male, the SV is approximately 70 ml and the left ventricular EDV is 120 ml, giving an ejection fraction of 70/120, or 0.58 (58%). An EF within the range of from 55-60% is considered normal. The ejection fraction of the right ventricle (“RVEF”) normally is equal to that of the left ventricle (“LVEF”) within narrow limits.
Echocardiography, radionuclide scanning [e.g., Multiple Gated Acquisition scan (MUGA), a nuclear scan that evaluates the pumping function of the ventricles, chambers and how the heart contracts] and left ventriculography were readily available and accurate measures of left ventricular ejection fraction (“LVEF”). Echocardiography has been utilized to determine end-systolic and end-diastolic volumes by using the biplane area length method.
Other measures of cardiac function in the post-infarct period included assessment of the stroke volume index and velocity of circumferential fiber shortening. Strauer, et al., Circulation 106: 1913-18 (2002). Stroke volume (SV) is the amount of blood the left ventricle ejects in one beat, measured in milliliters per beat (ml/beat). SV can be indexed to a patient's body size by dividing SV by Body Surface Area (BSA) to yield the Stroke Index (SI).
Assessment of repair of infarcted myocardium also included evaluation of peri-infarct region perfusion using thallium scintigraphy. Id.
Magnetic resonance imaging (MRI) is a useful tool for assessing cardiac function and viability (infarct size) in this setting. See Yin, A, et al., Blood 90: 5002-5012 (1997).
The day after successful stenting, subjects were assessed for study eligibility and, where appropriate, were offered informed consent to participate in the study. Subjects exhibiting symptoms for no more than three (3) days prior to successful stent placement were assessed, prior to discharge, for study eligibility. Subjects found to meet eligibility criteria (see infra) were offered informed consent to participate.
Consented subjects had a study entry echocardiogram no sooner than 96 hours after stent placement. Subjects were eligible to proceed on study if (i) the LVEF was less than or equal to 50% on echocardiography and (ii) a segmental ventricular wall abnormality was observed in the IRA.
Eligible subjects immediately completed baseline cardiac function and perfusion assessment. Specifically, baseline cardiac function included: transthoracic echocardiography at rest and with low dose dobutamine to assess cardiac function, including ejection fraction, end systolic and diastolic volumes, and wall motion score index and viability.
Echocardiography. Myocardial contrast echocardiography was used for on study screening four days after stenting to identify patients with left ventricular dysfunction (echocardiographic ejection fraction ≦50%).
Cardiac Perfusion. Perfusion was assessed using a routine Technetium (Tc-99m) Sestamibi radionuclide scan at rest and after intravenous adenosine at baseline and after 6 months. Perfusion defect size was estimated as the resting total severity score (RTSS) using resting single-photon emission computed tomography (SPECT). The Emory Cardiac Toolbox was used for image quantification; evaluation used a 17-segment model. A core review lab assessed the perfusion studies with the interpreter blinded to the study cohort. Improvements in perfusion were expressed in semi-quantitative terms (yes/no). The percentage of patients observed to have improvement in perfusion was compared between dose cohorts.
MRL. All enrolled subjects underwent gadolinium-enhanced cardiac magnetic resonance imaging (MRI) to evaluate left ventricular end systolic and diastolic volumes (LVESV and LVEDV), left ventricular ejection infraction (LVEF), and infarct size at baseline, 3 months, and 6 months. Subjects received Gadolinium contrast during scanning. MRI scan used the breath holding technique. Steady state precession imaging to obtain global and regional LV function was performed as was Gadolinium imaging. Left ventricular end systolic and diastolic volumes, LVEF, LV end diastolic dimension, wall thickness in systole and diastole of the infarcted region, and infarct size were reported using the AHA/AVV 17-segment model with transmural extent of the infarct reported as <25%, 26%-50%, 51%-75% and >76%. A core review laboratory assessed MRI with the interpreter blinded to the study cohort.
Subjects selected for this study must have met all of the following clinical criteria (“inclusion criteria”):

    
    
        Age: 18-75 years;
        Acute ST segment elevation myocardial infarction meeting ACC/AHA criteria, with symptoms of chest pain within 3 days of admission. These vriteria include (ST elevation >1 mm in limb leads or 2 min in two or more precordial leads and increased levels of troponin, creatine kinase MB (CPK MB) or both), New York Heart Association (NYHA) heart failure class (to be recorded) of I, II or III;
        Eligible for percutaneous coronary intervention (PCI);
        Eligible for MRI;
        Eligible for Single Proton Emission Computed Tomography (SPECT) imaging;
        Echocardiograph lab conclusion of ability to adequately assess cardiac parameters after review of admission echocardiography;
        Study entry echocardiogram [96 to 144 hours {i.e., about 4 days to about 6 days} after stent placement], LVEF less than or equal to 50% on echocardiography, and segmental ventricular wall abnormality in the IRA circulation by echocardiography after reperfusion;
        Subject must have been able to provide informed written consent and must have been willing to participate in all required study follow-up assessments;
        Subjects must have had a hemoglobin content (Hgb)>10 grams/dL, white blood cell count (WBC)>3500 cells/mm3, a platelet count >100,000 cells/mm3 and an international normalized ratio (INR, a blood coagulation test)<2.0 the day before the bone marrow collection;
        Subjects must have had a serum creatinine <2.5, total bilirubin <2.0 within 7 days of the bone marrow collection;
        IRA and target lesion must have been clearly identifiable when disease was present in more than one vessel;
        Successful reperfusion and intracoronary stent placement, with Thrombolysis In Myocardial Infarction (TIMI) 2 or 3 flow and IRA with <20% stenosis after revascularization;
        Subjects must have been deemed eligible to receive conscious sedation, mini-bone marrow harvest, and second catheterization for Chemotactic Hematopoietic Stem Cell Product infusion;
        The type of stent used and time and date inserted must have been recorded;
    
    


Drug eluting stents were limited to paclitaxel or sirolimus types;

    
    
        Included subjects must have had an expected survival of at least one year and must not have had multiple vessel disease after revascularization, or were not expected to require intervention within 6 months of study entry.
    
    


Subjects who satisfied any one of the following criteria did not qualify for, and were excluded from, the study (“exclusion criteria”):

    
    
        Subjects who were not candidates for percutaneous intervention, conscious sedation, MRI, SPECT imaging or mini-bone marrow harvest;
        History of sustained chest pain unrelieved by nitrates, occurring 4 or more days before revascularization;
        Subjects who failed to re-perfuse the infarct related coronary artery or to have successful stent placement;
        Echocardiography lab conclusion after admission echocardiography review that study was not adequate to assess cardiac parameters;
        Subjects presenting with cardiogenic shock (systolic pressure <80 on vasopressors or intra aortic counterpulsation);
        Subjects with a side branch of the target lesion >2 mm and with ostial narrowing >50% diameter stenosis after revascularization;
        Subjects unable to receive aspirin, clopidogrel or ticlopidine;
        Subjects receiving warfarin must have had an INR less than or equal to 2 [the term INR refers to INR International Normalized Ratio, which is a system established by the World Health Organization (WHO) and the International Committee on Thrombosis and Hemostasis for reporting the results of blood coagulation (clotting) tests];
        Subjects with severe aortic stenosis;
        Subjects with severe immunodeficiency states (e.g., AIDS);
        Subjects with cirrhosis requiring active medical management;
        Subjects with malignancy requiring active treatment (except basal cell skin cancer);
        Subjects with documented active alcohol and for other substance abuse;
        Females of child-bearing potential unless a pregnancy test was negative within 7 days of the mini-bone marrow harvest;
        Subjects with ejection fractions greater than 50% on study entry echocardiogram (96 to 144 hours after stent placement);
        Subjects with less than three months of planned anti-platelet therapy post index procedure;
        Subjects with multi-vessel disease after revascularization requiring subsequent planned intervention during the next 6 months;
        Subjects with participation in an ongoing investigational trial;
        Subjects with active bacterial infection requiring systemic antibiotics.
    
    


Baseline assessments of cardiac function and cardiac perfusion were obtained one day prior to the planned mini-bone marrow harvest and infusion of the chemotactic hematopoietic stem cell product (see infra). A mini-bone marrow harvest (“MMH”) was performed the day following baseline assessment of cardiac function and cardiac perfusion.
Example 2
Cardiac Catheterization
Sterile Preparation and Draping
Each subject was brought into the Cardiac Catheterization Laboratory after the investigator had obtained informed consent. The subject received a sterile preparation and draping in the Cardiac Catheterization Laboratory.
Cardiac Catheterization
Vascular access was obtained by standard technique using right or left groin. A sheath was placed in the femoral artery or the right or left brachial artery. Coronary arteriographic examination was performed by obtaining standard views of both right and left coronary arteries. Multiple views were obtained to identify the previously stented infarct related artery. All subjects received standard medications during the catheterization procedure in accordance with routine practice.
Example 3
Acquisition Process For Acquiring Chemotactic Hematopoietic Stem Cell Product that can then be Enriched for CD34+ Cells
While it is contemplated that any acquisition process appropriate for acquiring the chemotactic hematopoietic stem cell product comprising potent CD34+ cells is within the scope of the described invention, the following example illustrates one such process referred to herein as a mini-bone marrow harvest technique.
Preparation of Harvesting Syringes
Prior to the bone marrow harvest, forty 10 cc syringes loaded with about 2-ml of a preservative free heparinized saline solution (about 100 units/ml to about 125 units/ml, APP Cat. No. 42592B or equivalent) were prepared under sterile conditions. Heparin was injected via a sterile port into each of two 100-ml bags of sterile 0.9% normal saline solution (“Normal Saline”, Hospira Cat. No. 7983-09 or equivalent) following removal of 10 cc to 12.5 cc of normal saline from each bag, resulting in a final heparin concentration of about 100 units/ml (U/ml) to about 125 units/ml (U/ml). 2-ml of the preservative free heparin solution (about 100 U/ml to about 125 U/ml) were loaded under sterile conditions into each of the forty 10 cc syringes, which then were capped and placed into a sterile bag for transport to the harvesting site.
Subjects were prepared for bone marrow harvest after written informed consent was obtained as detailed in Example 1. Conscious sedation was provided using standard institutional procedures and guidelines. Bone marrow harvest was conducted under sterile conditions. The term “sterile conditions” as used herein includes proper scrubbing and gowning with a sterile mask and gloves worn by the harvesting attending and assistant.
The harvesting procedure, which can be performed outside of an operating room, was as follows: after sterile prepping and draping, each iliac crest was anaesthetized with a 1% lidocaine solution using a minimum of 10-ml for each crest. The area of anesthesia was a circular area no less than 10 cm in diameter. The harvesting needle was inserted until the iliac crest was punctured. The cap and stylet was removed and 2-ml of marrow was harvested into the 10-ml harvesting syringe containing 2-ml of the heparin solution. The syringe then was removed and placed on the sterile field. After re-inserting the stylet, the harvesting needle was advanced slightly and then rotated 90°. The stylet then was removed and an additional 2-ml of marrow was drawn into the harvesting syringe retrieved from the sterile field. This procedure was repeated two more times until the harvesting syringe contains 8-ml of marrow for a total of 10-ml of heparinized marrow at a final heparin concentration of about 20 U/ml to about 25 U/ml. Finally the full harvesting syringe was handed to the harvesting assistant and shaken and infused in the sterile collecting bag as described below. The harvesting physician then took the other harvesting needle that had been flushed previously with the heparin solution and repeated this process.
The full harvesting syringe was infused in the sterile collecting bag as follows. The harvesting assistant was handed the full harvesting syringe and emptied it in the 500-ml collecting bag though the sterile adaptor attached to the bag. Then the harvesting needle was flushed with the heparin solution in the flushing syringe and returned to the sterile field.
The harvesting process was repeated on one iliac crest until about 19 syringes had been collected and emptied in the collecting bag. The same process was repeated on the other iliac crest until another about 19 syringes have been filled. A total of thirty-eight 8 ml aspirations from both iliac crests (ideally 19 from each iliac crest) resulted in 302-ml of bone marrow harvested in a final volume of 380 ml at a heparin concentration of about 20 U/ml to about 25 U/ml.
The collecting bag was sealed by tying off the connecting tube three times and then clamped distal to the ties. The bag was appropriately labeled “Human Bone Marrow Collection” and the results of the harvesting procedure, including final volume collected and any procedure related complication, were recorded on the Mayo Clinical Risk Score (MCRS) case report form. The completed label was affixed to the bone marrow bag. The bag then was placed in a sterile carrying bag to be transported to the processing facility.
Example 4
Preparation of the Bone Marrow Product for Transportation
In one embodiment, the harvested bone marrow is transported to a processing facility as follows. When the clinical site is prepared to ship the bone marrow preparation, 24-hour notice is provided to the processing facility. The processing laboratory makes shipping arrangements at the earliest possible time for pickup for same day delivery to the processing laboratory. Immediately after the bone marrow is collected, the bone marrow product is placed in the supplied shipping container. The shipping container contains two small blocks of frozen wet ice on the bottom and a sheet of bubble wrap on top of the wet ice. The bone marrow product is placed into a secondary bag and the secondary bag is placed on top of the bubble wrap. A temperature tag monitor (a sensor used to monitor the internal temperature) is affixed to the interior of the box. Another layer of bubble wrap then is placed on top of the product before the shipping container is sealed off.
Example 5
Selection of CD34+ Cells from the Harvested Bone Marrow Product
CD34+ cells were isolated from the harvested bone marrow product. In one embodiment, CD34+ cells were isolated using the anti-CD34 monoclonal antibody (Mab), Dynabeads® M-450 Sheep anti-Mouse IgG, and PR34+TM Stem Cell Releasing Agent components of the Isolex 300i Magnetic Cell Selection System (Baxter Healthcare Corp. Cat. No. 4R9734) as described in U.S. Pat. Nos. 5,536,475, 5,035,994, 5,130,144, 4,965,204, 5,968,753, 6,017,719, 6,251,295, 5,980,887, 6,676,937, U.S. Published Application No. 2003/0232050, and the Isolex 300i Package Insert, each of which is incorporated herein by reference. This operating system was adapted for isolation of CD34+ cells from bone marrow according to the described invention.
Upon arrival at the processing laboratory, the harvested bone marrow product (in the collecting bag) was inspected immediately and the bag checked for any leakage. The collection should be free flowing with no apparent clumps and should not be hemolyzed. The collection was not used if the integrity of the bag had been breached in any way.
The bone marrow product was processed within about 12 hours to about 24 hours of inspection. A 300-ml or 400-ml transfer pack container was obtained, and a plasma transfer set was attached to the sampling port of the container. The bone marrow product was transferred from the collecting bag to the transfer pack container. The pooled bone marrow collection product was mixed thoroughly by inverting the container twenty (20) times.
The pooled bone marrow collection product then was sampled for analysis. According to one embodiment, a total volume of 2.0 ml of the product was removed and aliquoted as follows: 0.3 ml was used for a duplicate run of Complete Blood Count (CBC) using a hematology analyzer; 0.2-ml was dispensed into a 75×100-mm glass tube for the detection of Gram positive and Gram negative bacteria by Gram Stain (Gram Stain Kit, VWR, Cat. No. BB231401); as a sterility check, 0.6-ml was dispensed into a Tryptic Soy Broth (TSB) (VWR, Cat. No. 29446-184) bottle for aerobic bacteria growth assay, 0.6-ml was dispensed into a Fluid Thioglycollate Media (FTM) (VWR Cat. #29446-138) bottle for anaerobic bacteria growth assay, and 0.3-ml was used in flow analysis for CD34+ cell enumeration and cell viability.
The collection was weighed on an electronic scale, and the appropriate tare weight of the collection bag recorded. The relationship of the volume of the bone marrow product to the weight of the product can be expressed as

Volume (ml)=[Weight (gm) of product−Tare weight of bag (gm)]+1.06 (gm/ml)  (Formula 1)

The number of Total Nucleated Cells (TNC) in the bone marrow product was calculated using the white blood cell (WBC) count obtained from the CBC according to the following relationship:

TNC=WBC/μl×1000×Product volume (ml)  (Formula 2)

The number of CD34+ cells in the bone marrow product was calculated from the following relationship:

Total CD34+ cells in the bone marrow product=Number of CD34+ cell/μl×1,000×Product volume (ml)  (Formula 3)

The Red Blood Cell (RBC) volume of the bone marrow collection product was calculated from the following relationship:

RBC volume (ml)=Product volume (ml)×Hematocrit (%)/100  (Formula 4),

Following an initial calculation of the RBC volume, the bone marrow product is overwrapped and centrifuged at 100-g for 20 minutes at 20 C with brake setting “off”. After centrifugation, the bone marrow container carefully is removed from the centrifuge and hung within a Class 100 biological safety cabinet with the sampling ports facing downward. A plasma transfer set carefully is placed into the middle sample port of the bag, and the RBC fraction is withdrawn into a syringe. This procedure is repeated with additional, fresh syringes until the remaining RBC are removed from the MMH. The MMH is prepared from a washing procedure, which begins through centrifugation at 1,000-g for ten (10) minutes at 20° C. with the brake “off”<followed by plasma expression, all in preparation for a wash in a PBS-based Wash Buffer Solution [1% HSA and 0.41% sodium citrate (w/v) in PBS (Ca++ and Mg++ free)] prepared earlier in the process. Following addition of the Wash Buffer, the cells again are centrifuged at 1,000-g for ten (10) minutes at 20° C. with the brake in the “off” position. Following this (final) centrifugation, the cells are expressed using a plasma expressor, supernatant is removed, and 150-ml of PBS Wash Solution is transferred into the product bag using a 60-ml syringe. The packed cells are resuspended by manual massage. Following this wash procedure, the RBC depletion and nucleated cell (NC) recovery are calculated as follows. The TNC of the RBC depleted bone marrow product was determined from the relationship:

Total TNC of the RBC depleted product WBC/μl of RBC depleted product×1000×RBC-depleted MMH volume (ml)  (Formula 5)

The TNC recovery of the RBC depleted product, which must be at least 80% of the original product count, was calculated from the relationship:

TNC recovery=TNC of the RBC depleted product+TNC of the unprocessed product×100%  (Formula 6)

The total RBC volume was calculated as described supra; the RBC volume in the RBC depleted product should be ≦20-ml.
In accordance with one embodiment of the described invention, the Isolex 300i system was used to process the RBC-depleted product or the bone marrow product whose RBC volume was <20 ml according to the following processing steps:
(i) The bone marrow was washed automatically to remove platelets;
(ii) CD34 positive (CD34+) cells were labeled specifically for selection by incubation with the Isolex 300i CD34 monoclonal antibody (Mab);
(iii) Unbound reagent was removed by washing the cell suspension with buffer solution;
(iv) Sensitized CD34+ cells (meaning CD34+ cells labeled with CD34 Mab) were captured by Dynabeads M-450 Sheep anti-Mouse IgG;
(v) A selection column was used to separate the magnetically-labeled Dynabeads having captured CD34+ cells from unwanted cells, which were washed through the selection column and collected in the Negative Fraction Bag; and
(vi) PR34+ Stem Cell Releasing Agent released CD34+ cells from the column, and the CD34+ cells were collected in the End Product Bag. The system performed several washing steps, disposing of most of the liquid into the Buffer Waste Bag.
The Isolex® selected CD34+ fraction was assayed as follows to determine WBC and CD34+ cell yields. The volume of the CD34 Positive Fraction was determined by mixing the cells in the End Product Bag; the bag was gently massaged by hand to ensure even cell distribution. A transfer set was inserted into the sampling port of the End Product Bag and a 60-ml syringe was attached. The cell suspension was withdrawn into the syringe (maximum 50-ml at a time) in order to measure the total volume.
A 3-ml or 5-ml syringe was used to remove a 2.0-ml sample from the End Product Bag through the transfer set for quality control testing. The aliquoted volumes of the samples and the analyses performed on those samples were as previously described, i.e., CBC: 0.3-ml; Gram stain: 0.3-ml; CD34+ cell enumeration and cell viability: 0.2-ml.
The total TNC of the CD34 Positive Fraction was calculated from the relationship:

Total TNC of the Positive Fraction=WBC/μl of the Positive Fraction×1000×Volume of the Positive Fraction  (Formula 7)

The TNC recovery of the Positive Fraction, which must be less than 5% of the original product count, was calculated from the following relationship:

TNC recovery=Total TNC of the Positive Fraction+Total TNC of the unprocessed product×100%  (Formula 8)

The total number of viable CD34+ cells in the Positive Fraction was determined from the following relationship:

Total CD34+ cells in the Positive Fraction=Number of CD34+ cells/μl of the final product×1,000×Final product volume (ml)  (Formula 9)

The CD34+ cell recovery of the Positive Fraction was calculated from the following relationship:

CD34+ cell recovery=Total CD34+ cells of the Positive Fraction+Total CD34+ cells of the unprocessed product×100%  (Formula 10).

Example 6
Preparation of Selected CD34+ Cells for Transfusion
Samples of the chemotactic hematopoietic stem cell product were removed to be assayed for WBC count, by flow cytometry (for CD34+ cell enumeration and viability), Gram stain, and sterility.
CD34+ cells were characterized by flow cytometric analysis featuring CD34bright and CD45dim fluorescence by double labeling with anti-CD34 and anti-CD45 antibodies (Beckman Coulter, PN IM3630). CD34+ cells and CD45+ cell viability was determined by excluding the dying cells, which take up the intercalating DNA dye 7-aminoactinomycin D (7AAD). See Brocklebank A M, Sparrow R L. Cytometry. 2001; 46:254-261 (2001); Barnett D, et al. Br. J Haematol. 106:1059-1062 (1999); Sutherland, et al., J Hematotherapy 5:213-226 (1996), and U.S. Pat. Nos. 4,520,110; 4,859,582; 5,055,556; European Patent No. 76.695; Canadian Patent No. 1,179,942 (PE, APC); U.S. Pat. No. 4,876,190 (PerCP); U.S. Pat. Nos. 5,268,486; 5,486,616; 5,569,587; 5,569,766; 5,627,027 (Cy); U.S. Pat. Nos. 4,714,680; 4,965,204; 5,035,994 (CD34); U.S. Pat. No. 5,776,709 (Lyse/no-wash method); U.S. Pat. Nos. 5,723,218 and 5,187,288 (TruCOUNT Tubes), the contents of each of which is incorporated by reference herein in its entirety.
Any flow cytometer or an equivalent device can be used for conducting analysis of CD34+ cell enumeration and viability. In one embodiment, the processing laboratory employed a BD FACSCaliburTM flow cytometer and BD FACSCompTM software was used for instrument setup and monitoring. A template and a panel of legend labels were preinstalled for acquisition and analysis. Prior to use, the reagents, namely CD45FITC/CD34PE, Stem-Count Fluorospheres, Concentrated Ammonium Chloride Lysing Solution, and 7AAD Viability Dye, were brought to ambient temperature. CD34+ cell controls were run as a positive control to affirm that the instrument was set up for analyzing CD34+ cells, and the results were compared with the manufacturer's pre-determined CD34 percent range.
The unprocessed bone marrow product and Isolex® processed chemotactic hematopoietic stem cell products may be analyzed by many different procedures. In one embodiment, for example, immediately upon receiving the sample, if the WBC count of the sample is greater than 2×107 cells per ml, the sample is diluted with Sheath fluid to achieve a cell count of about 2×107 WBC per ml. 100 μl of the diluted product is aliquoted into two 15×100 mm tubes. Using a micropipetter, 20 μl of CD45FITC/CD34 PE and 7-AAD viability dye reagent are added into each tube and the samples gently vortexed. The tubes are covered with aluminum foil and left at ambient temperature for 15 to 20 minutes. RBCs are lysed by adding 1.5 ml of 1× Lysing Solution to each tube, vortexing gently. The tubes are incubated for ten minutes at ambient temperature, protected from light. The samples are stored at about 2° C. to about 8° C. (i.e., on an ice bath) protected from light until data acquisition is performed. Data acquisition must be performed within one hour of adding the lysing buffer. Before data acquisition, Stem-Count Fluorospheres are resuspended by end-over-end rotation (10 times). 100 μl of Fluorospheres is added to each tube and gently vortexed taking care not to generate air bubbles. The absolute count of CD34+ cells in the product is calculated from the relationship:

Number of viable CD34+ cells per μl of product=LCD34×FAC  (Formula 11)

where LCD34 is the averaged number of events for Live CD34+/All CD 45+; “FAC” is Fluorospheres Assayed Concentration; and F is the averaged number of Fluorosphere singlets counted.

The volume of CD34+Positive Fraction was calculated to obtain the number of CD34+ cells required for the required dosing. The Required Positive Fraction Volume (ml) is defined as:

The Requested CD34+ cell dosage+(Total CD34+ cells per μl in the Positive Fraction×1,000).  (Formula 12)

An appropriate number of cells was dispensed into a 50 ml conical tube and centrifuged at 500×g for 10 minutes. The supernatant was removed using a 30 ml serological pipette and disposed of as waste while exercising care not to disperse the cell pellets at the bottom of the tubes during this process. The infusion solution (20 ml) was added into the CD34+ Cell Positive Fraction tube and the cells dispersed using a 10 ml serological pipette by repeat pipetting. The resuspended cells were centrifuged for 10 minutes at 500 g. A 30 ml serological pipette was used (without disturbing the cell pellet) to transfer the supernatant/infusion solution into a 50 ml conical tube with a label “Positive Fraction Supernatant” affixed. The tube containing the supernatant was vortexed to homogenize the solution. A 10 ml serological pipette was used to transfer 10 ml of the homogenized supernatant back to the CD34+ Cell Positive Fraction tube. The remaining 10 ml of suspension in the Supernatant tube was used for sterility testing (5 ml each into a TSB (Trypticase Soy Broth) bottle and an FTM (Fluid Thioglycollate) bottle). The cells in the CD34+ Cell Positive Fraction were resuspended by slowly withdrawing and aspirating through a blunt end needle affixed to a 10 ml syringe (Infusion Syringe) several times. The cell suspension was withdrawn into the syringe, any air bubbles were aspirated off, and the blunt end needle removed. The infusion syringe was attached to the injection port of a 4-way stopcock.
The chemotactic hematopoietic stem cell product of the described invention was released for infusion only if it met the following criteria:

    
    
        CD34+ cell purity of at least about 70%, 75%, 80%, 85%, 90% or 95%;
        A negative Gram stain result for the selected positive fraction;
        Endotoxin Levels: less than about 0.5 endotoxin units/ml;
        Viable CD34+ cell yield of the “Chemotactic hematopoietic stem cell product” met the required dosing as per the treatment cohort;
        CD34+ cells were at least about 70%, 75%, 80%, 85%, 90% or 95% viable by 7-AAD;
        USP sterility result for “Positive Fraction Supernatant”: negative (14 days later); and
        Bone marrow CD34+ cell selection was initiated within about 12 hours to about 24 hours of completion of bone marrow harvest.
    
    


Sterility assessment on the stem cell product including gram staining and endotoxin was performed prior to product release for infusion. USP sterility (bacterial and fungal) culture was performed and the results were reported to the principal investigator. In the event of a positive USP sterility result, the subject and attending physician on call are notified immediately, provided with identification and sensitivity of the organism when available, and documentation of appropriate anti-microbial treatment and treatment outcome is recorded by the investigative site and the sponsor.
After meeting these release criteria, the chemotactic hematopoietic stem cell product was released for infusion and packaged for transportation to the catheterization facility. A sample also was sent for in vitro testing. Product was released only if CD34+ cell selection was initiated within 12 hours to about 24 hours of completion of bone marrow harvest and only if it was to be infused within about 48 hours to about 72 hours of completion of bone marrow harvest.
Example 7
Formulation of the Chemotactic Hematopoietic Stem Cell Product Comprising CD34+ Cells
The chemotactic hematopoietic stem cell product was formulated in 10-ml of saline (0.9% Sodium Chloride, Injection, USP, Hospira, Cat #7983-09) supplemented with 1% HSA (Human Albumin USP, Alpha, Cat. #521303) (“Infusion Solution”) and at least 20% autologous serum. In addition, there may be some trace amount of materials (quantities not determined) in the Chemotactic hematopoietic stem cell product that are used and left over during the product processing. These materials include: Dulbecco's. Phosphate. Buffered Saline-Ca++, Mg ++Free (D-PBS) (Baxter, Cat. # EDR9865), Sodium Citrate (Baxter/Fenwal, Cat. #4B7867), Hetastarch (Abbott Laboratories, Cat. #0074-7248-03), IVIg (Gammagard® Immune Globulin Intravenous, Baxter, Cat. #060384) and the reagents in the Isolex® 3001 Stem Cell Reagent Kit (Baxter, Cat. #4R9734) including anti-CD34 monoclonal antibody, stem cell releasing agent and Sheep anti-mouse magnetic beads.
Example 8
Transporting Chemotactic Hematopoietic Stem Cell Product to the Catheterization Facility
The chemotactic hematopoietic stem cell product that met the release criteria was loaded into a sterile 10 cc syringe in a Class 100 biological safety cabinet located within a controlled aseptic environment; e.g., at minimum, a Class 100,000 cell processing facility; class 10,000 is preferable, but not required. The chemotactic hematopoietic stem cell product was suspended in 10-ml PBS supplemented with HSA and the container labeled in accordance with release criteria. The clinical trial was designed to have four dosing cohorts consisting of five subjects each in each cohort. The first was to receive about 5×106 CD34+ cells, the second about 10×106 CD34+ cells, the third about 20×106 CD34+ cells and the fourth about 30×106 CD34+ cells. Subjects in cohorts 2-4 with inadequate CD34+ cell quantities to meet the assigned cohort dose were added to a prior cohort at the greatest possible CD34+ cell dose. The loaded infusion syringe was attached to a four-way stopcock along with a flushing syringe and capped; safety guards were applied to prevent leakage. The delivery apparatus was sealed in a double sterile bag and placed in a secure transportation box for transportation to the cardiac catheterization facility. Following release of the chemotactic hematopoietic stem cell product and cohort assignment, the chemotactic hematopoietic stem cell product was shipped to the catheterization site. In some embodiments, the chemotactic hematopoietic stem cell product was administered intravascularly, i.e., by direct infarct-related artery infusion. In some embodiments, the chemotactic hematopoietic stem cell product was administered parenterally into the myocardium.
Example 9
Intra-Coronary Infusion of Chemotactic Hematopoietic Stem Cell Product
Upon notification from the cell processing facility that the chemotactic hematopoietic stem cell product had been released for infusion (see supra), the subject/patient was scheduled to arrive at the catheterization facility at a time to coincide with the arrival of the chemotactic hematopoietic stem cell product.
Cardiac enzymes (brain natriuretic peptide (BNP), troponin and CPK MB), complete blood counts, a full chemistry panel (renal and liver function test) and an EKG were performed just prior to chemotactic hematopoietic stem cell product infusion. Clinical assessment of the stage of heart failure according to the New York Heart Association's (NYHA) functional classification system was recorded.
Upon receipt of the chemotactic hematopoietic stem cell product and final quality assurance release (by facsimile) for infusion, the subject underwent cardiac catheterization as detailed above. Coronary arteriography was performed to assess for patency (meaning openness, freedom from blockage) of the infarct related artery and Thrombolysis in Myocardial Infarction (TIMI) angiographic flow. A balloon catheter over a wire was placed in the stented segment of the infarct related artery. Any appropriate balloon dilatation catheter having an internal diameter of at least about 0.36 mm compatible with the chemotactic hematopoietic stem cell product infusion can be used. After positioning, the balloon wire was removed. The chemotactic hematopoietic stem cell product delivery apparatus was removed from the transportation case.
The delivery apparatus was in a sterile bag and had safety blocks attached to the infusion syringe (containing the chemotactic hematopoietic stem cell product) and the flushing syringe. The apparatus consisted of the infusion syringe (containing 10 ml of the chemotactic hematopoietic stem cell product) and the flushing syringe (containing 6 ml of flushing solution) wherein both were attached to a sterile four-way stopcock. The entire delivery apparatus was shaken gently to resuspend the CD34+ cells in the infusion solution. The flushing syringe was used to eliminate all air bubbles in the apparatus (to prevent air emboli) and the delivery apparatus then attached to the balloon dilatation catheter via the stopcock.
Delivery of the chemotactic hematopoietic stem cell product to the subject by infusion proceeded as follows. First, with the stopcock open between the flushing syringe (6 ml solution) and the central lumen of the balloon catheter, 1 ml of flushing solution was infused (after removal of the guard) into the central lumen of the catheter over 15 seconds. Second, the balloon was inflated at two atmospheres of pressure within the stent to avoid damage to the coronary artery endothelium and then the stopcock valve adjusted to allow infusion of the chemotactic hematopoietic stem cell product distal to the inflated balloon (after removal of the guard). With the balloon inflated, about 3 cc to about 4 cc from the infusion syringe was infused by hand over a period of about 30 seconds to about 45 seconds (to be timed and documented). The balloon remained inflated to allow adhesion of the CD34+ cells and to prevent back flow for a total of about 2 minutes to about 3 minutes (including the time for infusion). In between infusions, the balloon remained deflated for 3 minutes to allow restoration of blood flow (reperfusion). Generally, 3 infusions are required to empty the infusion syringe. Third, upon completion of infusing the chemotactic hematopoietic stem cell product and with the balloon deflated, the valve on the stopcock was adjusted to allow filling of the infusion syringe from the flushing syringe. Finally, with the balloon inflated (about 2 minutes to about 3 minutes), the 4 ml of flushing solution now in the infusion syringe was infused over a period of about 30 seconds to about 45 seconds to dislodge any residual CD34+ cells from the syringe and catheter into the IRA circulation. The catheter then was removed.
An infusion-related ischemia (inadequate blood flow) assessment was performed during the first 24 hours after chemotactic hematopoietic stem cell product infusion. An EKG at about 12 hours and at about 24 hours and analytical chemistry of cardiac enzymes (BNP, troponin and CPK MB) about every 8 hours for about 24 hours was obtained. Arrhythmia assessment (24 hour Holter monitor) was performed immediately post-chemotactic hematopoietic stem cell product infusion. Routine transthoracic echocardiography to evaluate global and regional left ventricular function was performed prior to the subjects' discharge after chemotactic hematopoietic stem cell product infusion.
Additional follow-up visits for safety assessments included visits at 1 week and 2 weeks after product administration. Visit assessments included a comprehensive medical history and physical examination, EKG, complete blood counts, full chemistry panel (renal and liver function test), and measure of serum cardiac markers (BNP, troponin and CPK MB). Clinical assessment of NYHA functional class was recorded on week 1 and 2.
At 4 weeks post chemotactic hematopoietic stem cell product infusion, an EKG and cardiac enzymes (BNP, troponin and CPK MB) was obtained. A 24 Holter monitor was used to assess for arrhythmias. Clinical assessment of NYHA functional class was recorded. Treadmill exercise testing using a symptom limiting Bruce protocol was performed as well.
At about 3 months and about 6 months post chemotactic hematopoietic stem cell product infusion, a 24 hour Halter monitor was performed. Clinical assessment of NYHA functional class was recorded. At about 6 months post-chemotactic hematopoietic stem cell product infusion, a symptom limited treadmill exercise testing using the Bruce protocol was recorded.
A safety assessment at about 12 months post-chemotactic hematopoietic stem cell product infusion includes a comprehensive medical history and physical examination, EKG, complete blood counts, full chemistry panel (renal and liver function test), and measure of serum cardiac markers (BNP, troponin and CPK MB). A 24 hour Holter monitor is performed, and clinical assessment of NYHA functional class is recorded.
Statistical Analysis
A paired design, where each subject serves as his or her own control, was used in some embodiments. Differences between before and after treatment, per subject, was analyzed for each of the four numeric cardiac functions (i.e., myocardial contractility; end systolic volume, end diastolic volume; and perfusion). Linear regression analysis was used to assess the significance of increased dosing levels. The null hypothesis is that the slope of the regression line (dosing level serving as the independent variable and the “after” minus the “before” difference serving as the dependant variable) is equal to zero. The power of rejecting a false null hypothesis is 0.68 at the 0.05 alpha level of significance for a high correlation of 0.5 between dosing and improvement in cardiac function. The 95% confidence interval about the slope of the regression line was used to assess the medical significance of the increase in dosing level. If the slope of the regression line was not significantly different from zero but the intercept of the regression line is different from zero, then all treatment groups were combined and a paired t-test performed to assess the overall treatment effectiveness. The null hypothesis is that the mean of the differences is equal to zero.
Baseline clinical and demographic characteristics were compared by treatment group using Student's t-test for continuous variables and chi-squared tests for categorical variables. Incidence rates for adverse events were compared among treatment groups using chi squared stest. For measurements of efficacy, pairwise differences from baseline to 6 month follow-up values in cardiac function and regional myocardial perfusion were compared between treatment and control groups using Student's t-test. Distributions of efficacy variables by treatment group were examined with box plot graphs. In post-hoc analysis, the influence of a dose threshold on treatment response was examined by combining control and 5 million cell cohort subjects and comparing them with 10 and 15 million cell cohort subjects using Student's t-test. Further the impact of cell characteristics on outcome measures was examined. For each of the pairs of variables of interest, the Pearson product-moment correlation coefficient (r) was calculated together with the corresponding p-value.
Type 1 (alpha) error was set at 0.05 for testing null hypothesis with tailed tst. Since analyses of secondary endpoints were exploratory in nature, no adjustment for multiple comparisons was made to the alpha errors reported. There were no imputations made to missing values in any variables analyzed and patients withdrawing from study were censored at the point of withdrawal. All statistical programs and analyses were conducted using SAS version 9.1 (Carey, N.C.).
A concurrent group (non-treated controls) meeting eligibility but not receiving CD34+ cells was evaluated similar to the treated group and assessed for significant improvement in cardiac function/perfusion. Each study site alternated accrual of treated and non-treated controls. A coin flip was used to determine the initial (treated or non-treated) subject sequence at each site. Comparison of outcomes between treated and non-treated groups was made. The core lab was blinded regarding treatment or no-treatment.
An assessment was performed to determine if a correlation existed between clinical outcome and cell content (CD34+) and/or in vitro colony growth (CFU-GM, CFU-GEMM, BFU-E), CXCR-4 mobility, and CXCR-4 and/or VEGF surface antigen expression. [See FIG. 4 and discussion below].
As planned, a total of 20 subjects were to receive the chemotactic hematopoietic cell product of the described invention. There was to be four dose cohorts (about 5×106, about 10×106, about 20×106, and about 30×106 CD34+ cells). If the chemotactic hematopoietic stem cell product content in any subject was not sufficient for the assigned cohort, that subject was reassigned to a prior cohort at the greatest possible dose. Subjects having fewer than 5×106 CD34+ cells available for infusion were removed from the study, did not undergo repeat catheterization and were not counted as part of the 20-subject study group. In addition, if the chemotactic hematopoietic cell product of the described invention did not meet release criteria, the subject did not receive the cell product and was not counted as a study candidate to be replaced by the next subject. In any cohort dosing group, if a subject experienced an acute (meaning immediate to about 7 days post infusion) unexpected toxicity considered to (probably) be a result of the cell product infusion, dose escalation was halted and 3 additional subjects were accrued to that dose level. If no other unexpected toxicity was observed, then dose escalation resumed; however the total of 20 subjects was not exceeded. If another toxicity occurred at that dose level, then all subsequent subjects were accrued to the next lower dose level.
The chemotactic hematopoietic stem cell product of the described invention was not administered to any subject in the higher dose cohort until all the subjects from the prior dose cohort had completed their follow-up assessments two weeks after product administration.
Example 10
Experimental Results of Preliminary Studies
A series of preliminary preclinical studies were performed in an attempt to accomplish the following goals:
(1) Optimize the manufacturing process for the Mini bone-Marrow Harvest (MMH);
(2) Evaluate the stability of the inbound MMH product and the outbound hematopoietic cell product;
(3) Evaluate the internal diameter allowance and safety of the catheters;
(4) Evaluate the compatibility of the cell product with the catheters intended to be used in the study; and
(5) Evaluate the suitability of using the supernatant of the final hematopoietic cell product to represent the final hematopoietic cell product for stability testing.
Study 1: Optimizing The Manufacturing Process for the Mini bone-Marrow Harvest (MMH)
The effect of key manufacturing variables on the yield of viable CD34 cells from representative bone marrow products was evaluated. A total of six (6) volunteer donors over the age of 45 (based on a range of 45-57) and three under 30 years of age (based a range of 21-28) agreed to donate an average of 45 ml (based on a range of 31 ml-54 ml) bone marrow and provided written Informed Consent for the procedure. The marrow aspiration technique employed was identical to that to be performed for the clinical scale MMH (see Example 3, supra). As shown in Table 2, the cell counts of nucleated cell (NC) and CD34+ cells of Mini bone-Marrow Harvest (“MMH”) derived cells collected from volunteer donors appeared to be age related.







TABLE 2







Effect of donor age on nucleated cell yield of the MMH.









Donor age group










Over 45 (45-57)
Under 30 (23-28)














Volume of
Viability
CD34 cells
Volume of MMH
Viability
CD34 cells


Donor
MMH (ml)
(%)
(105 per ml)
(ml)
(%)
(105 per ml)





1
31.30
83.85
1.27
48.00
96.90
7.98


2
43.50
97.42
3.89
50.60
96.28
11.60 


3
51.50
85.74
1.37
39.90
87.17
5.99


4
47.50
80.95
1.76
—
—
—


5
53.70
98.21
5.58
—
—
—


6
44.90
96.36
4.48
—
—
—


Avg.
45.40
90.42
3.06
46.17
93.45
8.52









The average cell count of the bone marrow products from older donors (N=6) was 28.4×106 (based on a range of 15.8×106-49.5×106) nucleated cells per ml [“NC/ml”], with an average viability, as determined by 7-AAD dye exclusion and flow cytometry, of 90.42% (based on a range of 80.95%-98.21%) and CD34+ content of 3.06×105/ml (based on a range of 1.27×105/ml-5.58×105/ml). In the younger subject group (N=3), the average cell count collected from marrow aspiration was 46.2×106 NC/ml (based on a range of 39.9×106 NC/ml-50.6×106 NC/ml), with an average 7-AAD viability of 93.5% (based on a range of 87.17%-96.90%) and total CD34+ content of 8.5×105/ml (based on a range of 5.99×105 CD34+ cells/ml-11.60×105 CD34+ cells/ml).
Red Cell Depletion and CD34 Selection







TABLE 3







CD34+ cell recovery after RBC depletion of MMH from


older age group (4557) donors.









Donor














1
2
3
4
5
Average

















Method of RBC
Hetastarch
Buffy
Buffy
Buffy
Buffy
—


depletion

coat
coat
coat
coat


CD34+ cell % in
1.09
1.64
1.63
1.45
1.99
1.58


MMH: Pre-RBC


depletion


CD34+ cell % in
1.33
1.55
1.51
1.61
1.84
1.57


MMH: Post-RBC


depletion


CD34+ cell recovery
65.68
92.36
80.66
78.79
81.67
79.83


post RBC depletion


(%)









As shown in Table 3, following red cell depletion of the MMH-derived bone marrow products collected from the older donors, an average of 79.83% (based on a range of 65.68%-92.36%) of the CD34 cells from the initial MMH was recovered. There was no significant difference between the initial CD34 cell purity (1.58%, based on a range of 1.09%-1.99%) and that following red cell depletion (1.57%, based on a range of 1.33%-1.84%).
Assay Method to Quantify Chemotaxis.
The assay used for the determination of in vitro migratory activity of CD34+ cells mediated by CXCR-4, which is adapted from an assay described in Jo et al (J. Clin. Invest. 105: 101-11 (2000)), relies on transmembrane migration of CD34+ cells. Transmembrane migration of CD34+ cells from the upper chamber to the lower chamber of a transwell polystyrene plate (6.5 mm diameter, 5 um pore size, Costar) is induced by SDF-1 placed in the lower chamber. The number of the migrated viable CD34+ cells in the lower chamber then is determined by flow cytometry analysis using CD34/CD45 antibodies and 7-AAD. Control spontaneous migration of CD34+ cells is performed without SDF-1 in the lower chamber.







TABLE 4







CD34+ cell recovery, purity, CXCR-4 migratory activity,


viability and hematopoietic CFU growth immediately after Isolex


processing of MMH from older age group (age 45-age 57) donors.









Donor














1
2
3
4
5
Average

















Storage time (hours) at
0
0
0
12
10.50
—


4° C.-8° C.


CD34+ cell
32.36
29.09
15.31
43.60
40.20
32.11


recovery (%)


CD34+ cell purity
76.76
73.64
71.66
72.52
72.01
73.32


(%}


CD34+ cell viability
98.49
93.80
97.38
98.28
98.39
97.27


CD34+ cell CXCR-4
22.10
2.60
22.00
19.90
19.70
17.26


migratory activity (%)


Hematopoietic CFU/100
27.5
25.0
18.9
17.0
21.00
21.9


CF34+ cells cultured









As shown in Table 4, following CD34 selection using the Isolex system, which includes immunomagnetic Dynabeads® and anti-CD34 mAb, an average of 32.11% (based on a range of 15.31%-43.60%) of the CD34 cells was recovered with an average purity of 73.32% (based on a range of 71.66%-73.64%) and an average viability of 97.27% (based on a range of 93.80%-98.49%). In addition, these CD34+ cells displayed an average of 17.26% (based on a range of 2.60%-22.10%) CXCR-4 migratory ability immediately after selection and were capable of generating hematopoietic colonies (21.89 colonies/100 CD34+ cells plated (based on a range of 17.0 colonies/100 CD34+ cells plated-27.5 colonies/100 CD34+ cells plated) in MethoCult culture.
Study 2: Evaluation of the Stability of the Inbound Mini-bone Marrow Harvest and of the Outbound Chemotactic Hematopoietic Cell Product.
A series of experiments, using healthy volunteers, was performed in order to evaluate the stability of the inbound MMH and of the outbound chemotactic hematopoietic stem cell product of the described invention. Assessment of the functional viability of the inbound and outbound products was evaluated by cell viability (7-AAD), SDF-1/CXCR-4 mediated CD34+ cell migration, and the ability to form hematopoietic colonies in methylcellulose (CFU colony forming ability).
To evaluate the inbound product stability for shipping and logistic purposes and for coordination with clinical schedules, MMH products were stored at 4° C. to 8° C. as indicated. To evaluate the outbound product stability for shipping and logistic purposes, the chemotactic hematopoietic stem cell product comprising isolated CD34+ cells enriched following MMH was stored at 4° C. to 8° C. as indicated.
In preliminary studies, cells either were processed immediately or maintained at 4-8° C. for 12 hours prior to processing to evaluate the impact of shipping and logistic duration on the manufacture of a suitable cell product for infusion. Despite the duration of storage prior to processing (inbound product expiration), the results did not vary significantly (data not shown).
In another series of experiments, cells were stored at about 4° C. to about 8° C. for 12 hours and about 24 hours prior to reassessment to simulate products infused at about 36 hours and at about 48 hours, respectively, following MMH.







TABLE 5







CD34+ cell viability, growth and CXCR-4 migratory


activity 13-13.5 hours after Isolex processing of MMH.









Donor











1
2
Average














CD34+ cell viability (%)
97.59
96.90
97.24


CD34+ cell CXCR-4 migratory activity (%)
7.70
7.50
7.60


Hematopoietic CFU/100 CD34+ cells cultured
18.00
25.00
21.5









As shown in Table 5, the isolated CD34+ cells of the chemotactic hematopoietic stem cell product had an average viability of 97.24% (based on a range of 96.90%-97.59%) and average CXCR-4-mediated migratory capacity of 7.60% (based on a range of 7.50%-7.70%). As shown in Table 6, after storage for an average of 26.3 hours (based on a range of 26.0 h-26.5 h), these cells had an average viability of 96.81% (based on a range of 96.39%-97.22%) and an average CXCR-4-mediated migratory capacity of 4.75% (based on a range of 4.50%-5.00%). Further, the cells still maintained their ability to generate hematopoietic colonies in vitro.







TABLE 6







CD34+ cell viability, growth and CXCR-4 migratory


activity 26.0-26.5 hours after Isolex processing of MMH.









Donor











1
2
Average














CD34+ cell viability (%)
97.22
96.39
96.81


CD34+ cell CXCR-4 migratory activity (%)
4.50
5.00
4.75


Hematopoietic CFU/100 CD34+ cells cultured
28.00
14.00
21.00









Thus, an average of 13.3 hours (based on a range of 13.0 h-13.5 h) after CD34+ cell selection, representing 26.0-26.5 hr post-MMH, the CD34+ cell population had an average viability of 97.24% (based on a range of 96.90%-97.59%), with average CXCR-4 mediated migratory capacity of 7.60% (based on a range of 7.50%-7.70%). At an average of 26.3 hours (based on a range of 26.0 h-26.5 h) following MMH, the average viability of the cells was 96.81% (based on a range of 96.39%-97.2%) and maintained an average CXCR-4-mediated migratory capacity of 4.75% (based on a range of 4.50%-5.00%).
Formulation of the composition of the described invention comprising this product occurred an average of 8 hours (8.63±1.80 N=4) hours after MMH collection, and infusion occurred within 24 hours of MMH.
In a subsequent experiment, four (4) MMH products (A-D) were collected and stored at 4° C. for an average of 12.8 hours (based on a range of 12.5 h-13.0 h) before the CD34+ cells were isolated by the Isolex procedure. This group, representing the “12 hour in-date” group (meaning that the product was formulated within the in-date time of about 12 hours), was evaluated for functional viability out-date at “24 hours” (22.9 h±1.63, N=4), “33 hours” (33.38±1.11, N=2), and “48 hours” (48.33±0.82, N=4) post MMH harvest. The data, summarized in Tables 7-9, demonstrate that following MMH, the chemotactic hematopoietic stem cell product comprising enriched CD34+ cells maintains 1) high viability (>90.0% average viability, Table 7), 2) 76.85% (±21.66) of their SDF-1/CXCR-4 mediated migratory ability (Table 8), and 3) their ability to form hematopoietic colonies in vitro (Table 9), respectively.







TABLE 7







CD34+ cell viability as a function of time after MMH:


12-hour in-dating and 48 hour outdating (all time points measured


from completion of MMH.).










Time (h)
CD34+ cell viability (%)














after MMH




Average



(SD)
A
B
C
D
(SD)



















98.22
97.13
97.60
99.00
97.99








(0.29)



24
95.32
97.76
—
—
96.54








(1.73)



33
91.92
96.32
95.90
80.00
91.04








(7.62)










Table 8 shows SDF-1/CXCR-4 mediated CD34+ cell migration (% migrating CD34+ cells) as a function of time after MMH: 12-hour in-dating and 48-hour outdating (all time points measured from completion of MMH). For the purpose of determining the impact of time post-MMH on the migratory ability of the CD34+ cells, time point “X” was considered the reference point, as this was determined to represent the earliest time point following MMH at which cells reasonably could be expected to be returned to the subject in a finished formulation. The remaining migratory activity at the following time points (Y=33 hours, Z=48 hours) was calculated as percent migratory ability remaining following the 24 hour (X) time point.







TABLE 8







SDF-1/CXCR-4 mediated CD34+ cell migration


(% migrating CD34+cells) as a function of time after MMH:


12-hour in-dating and 48-hour outdating (all time points


measured from completion of MMH).









Migrating CD34+ cells (%)

















Average


Time (h) after MMH
A
B
C
D
(SD)















24 (X)
20.00
18.50
21.50
36.00
24  







 (8.09)


% Remaining
100.00
100.00
100.00 
100.00 
100.00 







(0)  


33 (Y)
21.80
10.50
—
—
16.15







 (7.99)


*% Remaining
109.00
56.76
—
—
82.88







(36.94)


48 (Z)
8.80
17.00
17.50
31.00
18.58







 (9.19)


@% Remaining
44.00
91.89
81.40
86.00
75.85







(21.66)





*= (Y ÷ X) × 100%


@= (Z ÷ X) × 100%






Table 9 shows the number of colony forming units (CFU) per 100 viable CD34+ cells plated as a function of time after MMH: 12-hour in-dating and 48 hour-out-dating (all time points measured from completion of MMH).







TABLE 9







Number of colony forming units (CFU) per 100 viable CD34+


cells plated as a function of time after MMH









# of CFU per 100 viable



CD34+ cells plated














Time (h)




Average



after MMH
A
B
C
D
(SD)







24
13.00
30.00
37.00
39.00
29.75








(11.81)



33
12.00
34.00
—
—
23.00








(15.56)



48
15.00
30.00
20.00
 8.00
28.25








(14.57)










In an attempt to extend both the in-date and out-date stability parameters for the chemotactic hematopoietic stem cell product of the described invention comprising CD34+ cells from 12-hours (in-date) and from 48-hours (out-date) (12/48), respectively, to 24-hours (in-date) and 72-hours (outdate) (24/72), respectively, CD34 cells were purified about 12 hours after MMH harvest (12 hour in-date) and about 24 hours after MMH harvest (24 hour in-date) and analyzed for functional viability at about 48 hours and at about 72 hours total time from MMH to time of testing/anticipated infusion (48 hour out-date and 72 hour out-date, respectively). Specifically, the functional viability characteristics of two MMH/chemotactic hematopoietic stem cell products of the described invention were evaluated at 48 hours and 72 hours. The resulting data were further compared to the same indices derived at the previous 12/48 time points (Tables 7-9).
Tables 10-12 show that at 33 hours (based on 32.5±0.71, N=2), 48 hours (based on one data point at 49 hours), and at 72 hours (based on 72.5 h±0.71, N=2), the isolated CD34+ cells of the chemotactic hematopoietic stem cell product of the described invention maintained 1) over 90% viability (Table 10), 2) 102.19±32.69% of their SDF-1/VEGF/CXCR-4 mediated migratory ability (Table 11), and 3) their ability to generate hematopoietic colonies in vitro (Table 12).







TABLE 10







CD34+ cell viability as a function of time


after MMH: 24-h in-dating and 72-h outdating (all


time points measured from completion of MMH).









CD34+ cell viability (%)












Time (h) after


Average



MMH
A
B
(SD)
















33
98.00
99.00
98.50






(0.71)



48
—
97.00
97.00






(—)



72
91.00
97.00
94.00






(4.24)

















TABLE 11







SDF-1/CXCR-4 mediated CD34+ cell migration (% population of


migrated CD34+ cells as a function of time after MMH): 24-h in-dating


and 72-h outdating (all time points measured from completion of MMH).










Time (h) after
Migrating CD34+ cells (%)












MMH


Average



(SD)
A
B
(range)
















33
8.20
14.05
11.13






(2.93)



% Remaining
100.00
100.00
100.00






(0.00)



48
—
18.61
18.61






(—)



% Remaining
—
132.46
132.46






(—)



72
5.70
18.95
12.33






(6.63)



% Remaining
69.51
134.88
102.19






(32.69)










The % remaining ratios in Table 11 were determined as in Table 8 above.







TABLE 12







Number of CFU per 100 viable CD34+ cells plated as a function


of time after MMH: 24-h in-dating and 72-h outdating (all time


points measured from completion of MMH).









Time (h) after
# of CFU per 100 viable CD34+ cells plated











MMH


Average


(SD)
A
B
(range)





33
26.00
28.50
22.25





 (1.25)


48
—
16.80
16.80





(—)


72
14.50
27.50
21.00





(6.5)









Further evaluation of the functional viability parameters of the chemotactic hematopoietic stem cell product comprising isolated CD34+ cells of the described invention (“clinical product”) at 8 hours (8.6 h±1.80, N=4), 12 hours (12.87 h±1.92, N=4), 32 hours (one time point at 33.5 h), 48 hours (47.50 h±2.5, N=2), and 72 hours (71.5 h±0.50, N=2) after MMH shows that after 72 hours, the product retains its 1) viability (Table 13), 2) SDF-1/CXCR-4 mediated migratory ability (Table 14) and 3) ability to form hematopoietic colonies in vitro (Table 15), equivalent to the 24-hour time point.







TABLE 13







Clinical Product Experience: CD34+ cell viability


as a function of time after MMH.









CD34+ cell



viability (%)














Time (h)




Average



after MMH
A
B
C
D
(SD)


















8
98.30
99.08
90.00
96.45
95.96








(4.12)



12
98.89
96.96
99.00
99.43
98.57








(1.10)



33
—
93.42
—
—
93.42



48
—
93.15
91.58
—
92.37








(1.11)



72
—
91.25
89.25
—
90.30








(1.48)

















TABLE 14







Clinical Product Experience: SDF-1/CXCR-4 mediated CD34+ cell


migration (% migrating CD34+ cells as a function of time after MMH).









Migrating CD34+



cells (%)












Time (h)




Average


after MMH
A
B
C
D
(SD)















12 (X)
14.31
13.08
9.74
31.73
17.97







(11.34)


% Remaining
100.0
100.0
100.0
100.0
100.0 







(0)  


33 (Y)
—
6.17
—
—
 6.17


*% Remaining
—
47.17
—
—
47.17


48 (Y)
—
4.88
8.21
—
 6.55







 (2.35)


*% Remaining
—
37.30
84.29
—
60.79







(23.49)


72 (Y)
—
3.7
6.6
—
 5.15







 (2.05)


*% Remaining
—
28.29
21.19
—
24.74







 (3.55)





*= (Y ÷ X) × 100%






All remaining ratios were calculated as in Table 8 above.







TABLE 15







Clinical Product Experience: # of CFU per 100 viable CD34+


cells plated as a function of time after MMH.









# of CFU per 100 viable CD34+ cells plated












Time (h)




Average


after MMH
A
B
C
D
(SD)















12.
98.14
33.30
24.00
22.50
 44.49 (36.09)


33
—
16.50
—
—
16.5


48
—
19.56
20.50
—
20.03 (0.66)


72
—
20.45
21.19
—
20.82 (1.10)









Based on these data, extension of the in-dating to 24 hours (from 12-hours) and the out-dating to 72 hours (from 48 hours) for the CD34+ cell clinical product of the described invention is justified.
FIG. 1 indicates the equivalence of the functional viability of the chemotactic hematopoietic cell product of the described invention at 72 hours to the same indices evaluated at 48 hours.
Study 3: Catheter Safety.
The viability and potential efficacy of the chemotactic hematopoietic stem cell product of the described invention comprising potent CD34+ cells depends on the cells maintaining their potency as they pass through a catheter. The catheter used in the methods of the described invention has an internal diameter of at least 0.36 mm. Any type of catheter having an internal diameter of at least 0.36 mm may be effective in delivering the pharmaceutical compositions of the described invention.
In one embodiment, the catheter is a balloon catheter. Balloon catheter safety studies were conducted to determine whether high cell concentrations and repeated perfusions adversely affect cell viability, cell recovery or catheter integrity. Non-mobilized peripheral blood progenitors were used in order to obtain an adequate number of cells to perform the analysis. Catheters were assessed for infusion of the cell product of the described invention comprising selected CD34+ cells through the IRA. None of the 0.36 mm internal diameter catheters tested adversely affected CD34+ selected cell viability, growth in culture, or mobility in CXCR-4 assays.







TABLE 16







Viability of CD34+ cells before and after


infusions through the catheters.









Viability (%)













Catheter
Condition
1
2
3
4
5












—
Pre-infusion
81.45













Raptor
After 1st infusion
84.29
70.94
87.89
88.02
84.68



After 2nd infusion
83.00
87.44
86.39
79.91
83.18


Sprinter
After 1st infusion
93.39
91.09
84.13
88.28
81.68



After 2nd infusion
91.89
91.08
84.88
77.65
77.73


Voyager
After 1st infusion
94.21
86.21
83.08
77.53
69.68



After 2nd infusion
88.03
84.71
79.27
78.11
76.80


Maverick
After 1st infusion
90.00
89.76
90.79
85.49
81.31



After 2nd infusion
90.94
87.38
81.98
80.09
85.47









As shown in Table 16, in all catheters tested, average CD34+ cell viability was at or above 70% following passage through the catheters.
To demonstrate that infusion of the CD34+ cell product does not pose any safety breach of the catheter used and that a significant percentage of cell product does not adhere to the interior walls of the catheter, catheters were challenged with repeat infusions of a CD34+ cell product having a considerably higher cell concentration than that used clinically. Four brands of catheters (Sprinter, Voyager, Maverick and Raptor) were evaluated using 5 catheters of each type. Non-mobilized apheresis products were used in order to obtain an adequate number of cells to perform the analysis. A cell concentration greater than three times that planned as treatment doses for the trial, i.e., 160×106 nucleated cells containing CD34+ cells in 10 ml of infusion solution, was passed twice through each catheter. The average CD34+ cell recovery was 100.59% (based on a range of 76.99% to 228.70%) following passage through the catheters.
All twenty catheters were tested for integrity using a methylene blue dye leak test after two perfusions with the nucleated cells. There was no evidence of leakage and the contact points and catheter tips were normal upon inspection.
As shown in Tables 17a and 17b, the effect on the cells of their perfusion through a catheter appears to be independent of catheter model and make among those catheters tested and was independent of the amount of time the cells were stored either prior to processing and/or after CD34+ cell selection and prior to perfusion, resulting in a final formulation containing an average recovery of 96.0% (range 80.8%-102.2%) of the CD34+ cells (Table 17b) and 86.36% of the CD45+ cells perfused through the catheter. Further, the average viability of the cells was 96.5% (range 92.5%-98.6%, N=16); the cells maintained both CXCR-4 migratory capacity (data not shown) and their ability to form hematopoietic colonies in methylcellulose (average 25.8 CFU/100 cells seeded (range 21.0%-30.5%)







TABLE 17a





CD45 cell recovery and viability after being infused through the catheters.



















1
2
3














Catheter
Condition
Recovery
R'd viab
Recovery
R'd viab
Recovery
R'd viab





Raptor
After 1st
69.68%
−1.35%
78.67%
2.08%
72.14%
−4.55%



infusion



After 2nd
97.91%
−8.55%
81.84%
−4.76%
142.98%
3.28%



infusion


Sprinter
After 1st
76.74%
−0.60%
68.56%
4.01%
72.63%
5.29%



infusion



After 2nd
78.82%
2.86%
85.40%
0.98%
90.29%
−1.02%



infusion


Voyager
After 1st
87.38%
1.58%
83.93%
−0.36%
103.58%
0.93%



infusion



After 2nd
82.70%
7.01%
69.34%
15.90%
69.54%
10.40%



infusion


Maverick
After 1st
73.97%
1.58%
87.01%
0.42%
78.31%
0.69%



infusion



after 2nd
152.35%
−5.06%
73.44%
2.78%
80.85%
−3.92%



infusion














4
5
Average (1 to 5)














Catheter
Condition
Recovery
R'd viab
Recovery
R'd viab
Recovery
R'd viab





Raptor
After 1st
80.54%
1.83%
73.21%
−2.13%
74.85%
−0.82%



infusion




(30.83%)
(2.53%)



After 2nd
107.82%
−8.48%
94.08%
0.08%
104.93%
−3.69%



infusion




(47.60%)
(4.94%)


Sprinter
After 1st
73.61%
6.06%
66.83%
8.31%
71.67%
4.61%



infusion




(29.48%)
(3.51%)



After 2nd
82.22%
6.50%
91.61%
0.00%
85.67%
1.86%



infusion




(35.30%)
(2.76%)


Voyager
After 1st
95.82%
4.52%
131.55%
−4.39%
100.45
0.46%



infusion




(44.39%)
(2.91%)



After 2nd
89.04%
0.27%
69.03%
7.50%
75.93%
8.22%



infusion




(32.11%)
(6.09%)


Maverick
After 1st
75.53%
2.61%
77.22%
2.95%
78.41%
1.65%



infusion




(32.33%)
(1.21%)



After 2nd
97.10%
−2.97%
91.11%
−2.07%
98.97%
−2.25%



infusion




(49.11%)
(2.85%)











Average of all
86.36%
1.26%



catheters







aRecovery of CD45+ cells = (# of CD45 cells after infusion ÷ # of CD45 before infusion) × 100%



bReduction of CD45+ cell viability = [1 − (CD45+ cell viability % after infusion ÷ CD45+ cell viability % before infusion)] × 100%













TABLE 17b





CD34 cell recovery and viability after being infused through the catheters



















1
2
3














Catheter
Condition
Recovery
R'd viab
Recovery
R'd viab
Recovery
R'd viab





Raptor
After 1st
116.49%
−3.48%
121.62%
12.91%
110.89%
−7.91%



infusion



After 2nd
91.66%
1.53%
85.18%
−23.26%
122.47%
1.71%



infusion


Sprinter
After 1st
89.19%
−14.66%
83.34%
−11.83%
102.72%
−3.29%



infusion



After 2nd
103.52%
1.61%
99.82%
0.01%
82.11%
−0.89%



infusion


Voyager
After 1st
81.02%
−15.67%
96.08%
−5.84%
90.16%
−2.00%



infusion



After 2nd
106.48%
6.56%
81.66%
1.74%
95.04%
4.58%



infusion


Maverick
After 1st
76.99%
−10.50%
101.79%
−10.21%
98.62%
−11.46%



infusion



After 2nd
228.70%
−1.05%
88.66%
2.65%
103.35%
9.70%



infusion














4
5
Average (1 to 5)














Catheter
Condition
Recovery
R'd viab
Recovery
R'd viab
Recovery
R'd viab





Raptor
After 1st
97.55%
−8.06%
96.14%
−3.97%
108.54%
−2.10%



infusion




(45.46%)
(7.79%)



After 2nd
111.33%
9.21%
98.96%
1.78%
101.92%
−1.81%



infusion




(43.73%)
(11.14%)


Sprinter
After 1st
84.57%
−8.39%
88.65%
−0.28%
89.69%
−7.69%



infusion




(37.26%)
(6.16%)



After 2nd
114.87%
12.05%
100.45%
4.84%
100.15%
3.52%



infusion




(42.22%)
(4.90%)


Voyager
After 1st
82.73%
4.82%
89.32%
14.46%
87.86%
−0.85%



infusion




(36.28%)
(10.13%)



After 2nd
94.81%
−0.75%
91.01%
−10.23%
93.80%
0.38%



infusion




(39.12%)
(5.86%)


Maverick
After 1st
112.58%
−4.96%
96.05%
0.18%
97.21%
−7.39%



infusion




(41.34%)
(5.34%)



After 2nd
89.35%
6.31%
117.63%
−5.12%
125.54%
2.50%



infusion




(73.48%)
(5.33%)











Average of all
100.59%
−1.68%



catheters







aRecovery of CD34+ cells = (# of CD34 cells after infusion ÷ # of CD34 before infusion) × 100%



bReduction of CD34+ cell viability = [1 − (CD34+ cell viability % after infusion ÷ CD34+ cell viability % before infusion)] × 100%






Collectively these experiments demonstrate that the serial passage of a chemotactic hematopoietic stem cell product comprising CD34+ cells through a cardiac catheter with an internal diameter of at least about 0.36 mm does not adversely affect either catheter integrity or CD34+ cell potency, i.e., CD34+ cell viability, CFU colony growth, or CD34+CXCR+ mediated migratory capacity/mobility.
Study 4: Compatibility of the Cell Product with the Catheters
To further test the compatibility of the chemotactic hematopoietic stem cell product comprising CD34+ cells with each of the catheters that may be used for delivery of the cell product in the study, cell products were tested after multiple passages through each catheter type to evaluate the effects of extreme conditions of stress that would be greater than those expected during the treatment protocol.
At 48 hours post-MMH harvest, the chemotactic hematopoietic stem cell product comprising a range of about 5.73×106 CD34+ cells to about 21.10×106 CD34+ cells (i.e., dosages reflective of the treatment cohort) obtained from individual donors was infused sequentially through three catheters of the same brand, one type of catheter for each donor (Sprinter, Voyager or Maverick), and the cell product assessed for CD34+ cell recovery, colony formation and viability.
As shown in Table 18, viable, colony forming cells were recovered in all experiments for all three catheters tested (cell recovery 99%, 99% and 106%).







TABLE 18







CD34+ cell recovery and sterility after


sequential infusions through the catheters.









Catheter used











Condition
Parameter
Sprinter
Voyager
Maverick





Pre-infusion
CD34+ cell yield
9.72 × 106
2.11 × 107
5.73 × 106


After 1st
CD34+ cell recovery
111% 
103%
99%


catheter


After 2nd
CD34+ cell recovery
94%
104%
97%


catheter


After 3rd
CD34+ cell recovery
99%
 99%
106% 


catheter



Sterility (aerobic and
Negative
Negative
Negative



anaerobic microbes)









As shown in Table 19, the average viability of the CD34+ cells after passing through the third catheter was 94.000% (based on a range of 93.55%-94.40%) versus 96.01% (based on range of 94.18%-97.93%) of the pre-infusion cell product.







TABLE 19







CD34+ cell viability after sequential


infusions through the catheters.









CD34+ cell viability











Condition
Sprinter
Voyager
Maverick
Average





Pre-infusion
94.18%
95.91%
97.93%
96.01%


After 1st catheter
94.73%
96.31%
95.45%
95.50%


After 2nd Catheter
95.34%
95.72%
95.01%
95.36%


After 3rd catheter
93.55%
94.40%
94.04%
94.00%









As shown in Table 20, colony forming unit (CFU) growth derived from the CD34+ cells after passing through the third catheter was 95.27% (based on a range of 43.47%-163.64%) of the infusion product (i.e., the infused chemotactic hematopoietic stem cell product comprising CD34+ cells).







TABLE 20







CFU growth of CD34+ cells after sequential


infusions through the catheters.









CFU per 100 CD34+ cells cultured










Condition
Sprinter
Voyager
Maverick













Pre-infusion
30.5
11.5
11.0


After 1st catheter
22.0
14.0
22.0


After 2nd catheter
20.5
4.0
19.0


After 3rd catheter
24.0
5.0
18.0


Recovery from the pre-
78.69%
43.47%
163.64%


infused product after the 3rd


catheter








Average recovery
95.27%









To determine the effect of catheter perfusion on CD34+ cell mobility and ability to grow in culture, a series of experiments were performed where MMH cells obtained from healthy donors were stored at 4° C. for 12 or 24 hours before initiation of Isolex processing. Isolated CD34+ cell product that had been stored for about 12 hours pre-Isolex processing then were stored at 4° C. until about 36 hours had elapsed from the end of processing, for a total of about 48 hours post MMH. At that time they were assessed for SDF-1/CXCR4 mobility and CFU growth pre and post perfusion through a 0.36 mm inner diameter (i.d.) cardiac balloon catheter. Similarly, cells that were stored pre-Isolex processing for 24 hours then were stored at 4° C. until 48 hours had elapsed from the end of Isolex processing, for a total of 72 hours, and then assessed.
The results in Table 21 demonstrate that neither CD34+CXCR-4-mediated cell mobility nor the cell's ability to grow in culture at any of the time points tested was affected adversely by perfusion through a catheter having an internal diameter of at least 0.36 mm.







TABLE 21







12 inbound/48 outbound and 48 hour inbound/72 hour outbound from


MMH: SDF-1/CXCR4 mobility (% population of migrated


CD34+ cells) and CFU (per 100 viable CD34+ plated)


pre catheter perfusion (“PRE”) and post catheter


perfusion (“POST”).








Time (h) after



MMH
SDF-1/CXCR4 mobility (%) // # of CFU per 100 viable


Inbound/
CD34+ cells plated












outbound
A
B
C
D
E





12/48
2.7 // 14
 8.8 // 15
15.8 // 16
—
—


PRE


12/48
3.4 // 15
18.9 // 13
17.6 // 8 
—
—


POST


24/72
—
—
—
34 // 37
18.9 // 27.5


PRE


24/72



34 // 43
23.5 // 24  


POST









The Stabilizing Effect of Serum
The following data confirm the importance of the stabilizing effect of serum to the migratory capability of the selected CD34+ cells.
As shown in Table 22, no CXCR-4 migratory activity was observed for all samples tested including the pre-catheter infusion samples when the composition comprising a chemotactic hematopoietic stem cell product was formulated without serum.







TABLE 22







Chemotaxis of CD34+ cells after sequential infusions


through the catheters in the absence of serum.









Migration (%)












Condition
Sprinter
Voyager
Maverick







Pre-infusion
0.0
0.0
0.1



After 1st catheter
0.0
0.0
0.0



After 2nd catheter
0.0
0.0
0.1



After 3rd catheter
0.0
0.0
0.0










FIGS. 2 and 3 further illustrate that Isolex selected CD34+ cells retain their migratory capacity longer when formulated in the presence of human serum. Following Isolex processing, the bone marrow derived hematopoietic stem cell product comprising selected CD34+ cells was formulated either in (1) phosphate buffered saline (Dulbecco's phosphate buffered saline, Ca++, Mg++ Free (Baxter Cat. No. EDR9865) (“PBS”) containing 1% human serum albumin, 25 U/ml of heparin sodium and various concentrations (about 0%, about 10%, about 20%, or about 70%) of autologous serum; or (2) normal saline (0.9%) containing 1% human serum albumin, 25 U/ml of heparin sodium and (about 0% or about 10%) autologous serum. SDF-1/CXCR-4 mediated CD34+ cell migratory capacity was evaluated at different times during final product storage (at 2° C.-8° C.) and after passing the cells through the catheter at the same rate and duration as anticipated by the clinical protocol. None of these formulations affected CD34+ cell viability or the recovery of CD34+ cells after they had been passed through the catheter.
Regardless of whether the chemotactic hematopoietic cell products comprising selected CD34+ cells was (i) formulated either in PBS-serum or in saline-serum and (ii) either passed through the catheter immediately or passed through the catheter after a prolonged stability testing storage interval at about 4° C. to about 8° C., they maintained an average of 96.6% viability (range 92.5%-98.6%) and an average CXCR-4-mediated migratory capacity of 11.4% (range 2.4%-30.6%), representing a total time from harvest to mobility analysis of up to 48 hours.
As shown in FIG. 2 panel (a), cells formulated in PBS alone at about 25 hours retained about 10% of their CXCR-4 migratory capacity, which dropped off to near 0 at about 48 hours. As shown in panel (b), cells formulated in normal saline alone retained little, if any, of their migratory capacity. As shown in panels (c) and (d), cells formulated with PBS containing at least about 10% serum retained about 10-15% of their migratory capacity for up to about 55 hours (c), while cells formulated with saline and at least about 10% serum retained about 20% of their migratory capacity for up to about 50 hours. As shown in panels (e) and (f), cells retained a higher migratory capacity for a longer duration in PBS supplemented with even higher concentrations of serum.
As shown in FIG. 3, the product of the described invention comprising selected CD34+ cells when formulated in 10% serum, retained 14.25%, <1%, 6%, and 5.8% of its CD34+CXCR4-mediated migratory capacity about 24, about 32, about 48 and about 56 hours after harvest, respectively. FIG. 3 further shows that the product of the described invention comprising selected CD34+ cells when formulated in 20% serum retained 18.25%, 10.25%, 17% and 11% of its CD34+-CXCR4-mediated migratory capacity about 24, about 32, about 48 and about 56 hours after harvest, respectively. The term “stabilizing amount” as used herein therefore refers to the amount of serum that, when included in the formulation of the product of the described invention comprising selected CD34+ cells, enables these cells to retain their CXCR-4 mediated chemotactic activity and hematopoietic colony forming ability.
Study 5: Final Product Sterility Testing
Due to the limited yield of CD34+ cells obtained from a 300-ml MMH, final cell product sterility is assessed using the supernatant removed from the final product formulation in order to preserve cell product for infusion. Supernatant samples are loaded into the syringes in a manner identical to that used to load the cell product into the syringes used for infusion (see supra).
To demonstrate that such a sample is representative of the final cell product formulation, we inoculated selected CD34+ cells in infusion solution prior to centrifugation of the final product with C. sporogenes (13 CFU/ml), P. aeruginosa (2 CFU/ml), S. aureus (18 CFU/ml), A. niger (17 CFU/ml), C. albicans (3 CFU/ml) and B. subtilis (17 CFU/ml) (See Table 23). After centrifugation, the sterility of both cell pellet and non-cell supernatant fractions was assessed using USP aerobic and anaerobic testing.







TABLE 23







Bacteria and fungi used for the sterility study.













Expected CFU/ml of



Total # of
Total
inoculated


Microbe
microbes/ml
CFU/ml
sample (21 ml)













C. sporogenes
400
279
13


P. aeruginosa
400
36
2


S. aureus
400
371
18


A. niger
400
356
17


C. albicans
400
62
3


B. subtilis
400
349
17





Each source microorganism vial prepared by Microbiological Environments contained 400 microbes per ml, but the numbers of CFU derived from each species are varied.






As shown in Table 24, both the cell pellet fraction and suspension fractions from all tested samples showed outgrowth of the inoculated microorganisms, while un-inoculated controls showed no growth. Further, no apparent differential growth rate was observed between testing of cell pellet fractions and the suspension fractions for all microorganisms tested. Samples taken before each step of the processing procedure and following the final perfusion through the catheters all tested negative for microbial contamination.







TABLE 24







14-day sterility testing of nucleated cell (NC) samples inoculated with


specific species of microorganism (400 microbes in 21-ml NC sample).












Sample with microbe
Medium






Inoculated
type
Sample fraction
Test 1
Test 2
Test 3





C. sporogenes
FTMa
Cell pellet
Positive
Positive
Positive




Suspension
Positive
Positive
Positive


S. aureus
FTM
Cell pellet
Positive
Positive
Positive




Suspension
Positive
Positive
Positive


P. aeruginosa
FTM
Cell pellet
Positive
Positive
Positive




Suspension
Positive
Positive
Positive


A. niger
TSBb
Cell pellet
Positive
Positive
Positive




Suspension
Positive
Positive
Positive


C. albicans
TSB
Cell pellet
Positive
Positive
Positive




Suspension
Positive
Positive
Positive


B. subtilis
TSB
Cell pellet
Positive
Positive
Positive




Suspension
Positive
Positive
Positive


Positive control: C. sporogenes
FTM
Cell
Positive


Positive control: S. aureus
FTM
suspension
Positive


Positive control: P. aeruginosa
FTM

Positive


Positive control: A. niger
TSB

Positive


Positive control: C. albicans
TSB

Positive


Positive control: B. subtilis
TSB

Positive


Negative control: No microbes
FTM
Cell
Negative


Negative control: No microbes
TSB
suspension
Negative





aFluid thioglycollate medium


bTryptic soy broth






Preclinical Study Summary
Collectively, these preclinical data indicate that the manufacturing and testing procedures described are capable of generating adequate numbers of viable cells with adequate stability to withstand shipment and perfusion through the catheter in a manner that should pose no additional safety concerns to the subject other than those associated with the routine use of fluid infusion through the balloon catheter.
Example 11
Phase 1 Efficacy Data
The following phase I efficacy data show that within ≧10×106 isolated CD34+ cells, there are enough potent cells expressing CXCR-4 and having CXCR-4-mediated chemotactic activity to effect a biologic effect (paracrine and neoangiogenic), which prevents cardiomyocyte cell death and later changes consistent with ventricular remodeling.
In accordance with the disclosure in Example 1, a total of 31 subjects were consented, eligible and enrolled in the study. The 31 patients enrolled in the phase I study were randomly assigned to an autologous stem cell harvest treatment group or to a control group five days after an ST elevation myocardial infarction (STEM) characterized by a prolonged period of hypoperfusion (meaning blocked blood supply) Of the 31 subjects enrolled, 16 were in the treatment group and 15 in the control group. The first subject at each Center was randomized to either treatment or control, and each subsequent patient was enrolled into alternating treatment or control groups. if the subject was assigned to treatment, they continued into the Treatment Phase as long as all inclusion/exclusion criteria continued to be met. Subjects assigned to the control group progressed to the follow-up phase. There were no significant differences between groups in any of the baseline demographic or clinical characteristics. Patients enrolled were from 34 to 71 years of age, 87% male, 77% white, 61% in NYHA Class II or III and 49% in NYHA Class I, 74% experienced an infarcted left anterior descending coronary artery, and 55% received a drug eluting stent. LVEFs obtained on screening echocardiography varied between 25% and 50%.
CD34+ cells were isolated from the bone marrow by the mini-bone marrow harvest procedure as described in Example 3 within 5-8 days post stent replacement. Harvested marrow then was shipped to the cGMP cell processing facility as described in Example 4 and isolated as described in Example 5.
As originally planned, and as described in Example 8, there were to be four dosing cohorts (5 Million, 10 million, 15 million and 20 million CD34+ cells) in the study. However more than 15 million cells post CD34+ selection could not reliably be obtained. Therefore enrollment terminated at the end of cohort 3 with 15×106 being the highest cell dose assessed.
Following cell product release and cohort assignment, the CD34+ cell product was shipped to the catheterization site for direct infarct related artery infusion. Treatment infusion occurred 6-9 days post stent replacement (and within 48 hours of mini-bone marrow harvest). Subjects were brought to the catheterization laboratory only after the CD34+ cell product had arrived at the facility and had received final release for infusion.
The dosing cohorts consisted of 5 subjects in cohorts 1 and 2, 6 subjects in cohort 3, and 15 control subjects. For cohort 1, the chemotactic hematopoietic stem cell product of the invention comprised 5×106 isolated CD34+ hematopoietic stem cells containing a subpopulation of at least 0.5×106 potent CD34+ cells expressing CXCR-4 and having CXCR-4 mediated chemotactic activity [represented as “5 M”}. For cohort 2, the chemotactic hematopoietic stem cell product of the invention comprised 10×106 isolated CD34+ hematopoietic stem cells containing a subpopulation of at least 0.5×106 potent CD34+ cells expressing CXCR-4 and having CXCR-4 mediated chemotactic activity [represented as “10 M”]. For cohort 3, the chemotactic hematopoietic stem cell product of the invention comprised 15×106 isolated CD34+ hematopoietic stem cells containing a subpopulation of at least 0.5×106 potent CD34+ cells expressing CXCR-4 and having CXCR-4 mediated chemotactic activity [represented as “15 M”}. Control subjects (i.e., those not receiving CD34+ cell infusion) were not expected to have significant improvements in cardiac function (ejection fraction, end systolic and diastolic volumes, ventricular wall motion score index), or infarct region perfusion at 6 months follow up.
The subpopulation of potent cells that (i) express CXCR-4 and (ii) have CXCR-4 mediated chemotactic activity, expressed VEGFR-2 at very low levels (mean 0.84%, range 0 to 2.39%). Because the subpopulation of potent CD34+ cells co-expresses CXCR-4, {CXCR-4 co-expression; mean 60.63%, median 52% range 31-98% of CD34+ cells, capable of migrating in an SDF-1 gradient} while less than 2.5% of the CD34+ cells co-expresses VEGFR-2, functionally, these cells are VEGFR-2-, i.e., VEGFR-2 is not what is driving the cells into the peri-infarct zone.
A sterile pharmaceutical composition of the described invention was delivered to each subject in cohorts 1, 2, and 3 parenterally by infusion via the infarct-related artery through a catheter seven to eleven days following the STEMI as described in Example 9. The sterile pharmaceutical composition comprised: (a) a therapeutically effective amount of a sterile chemotactic hematopoietic stem cell product, the chemotactic hematopoietic stem cell product comprising an enriched population of isolated CD34+ cells containing a subpopulation of potent cells expressing CXCR-4 and having CXCR-4-mediated chemotactic activity; which, when passed through the catheter remained potent, and (b) a stabilizing amount of serum.
Cardiac function follow-up was performed at 3 and 6 months post-infusion. Cardiac infarct region perfusion was assessed at 6 months post infusion. Both perfusion and functional follow-up testing was assessed by a core lab facility blinded to the study treatment status of each subject. Comparison of these results to baseline indices was performed. Long term follow-up visits are conducted at 12 months and telephone interview with subjects will be made annually at years 2 through 5. On the 12-month follow-up visit echocardiograms, a number of the subjects had segmental wall motion abnormalities, not uncommon in this patient population, and none of the subjects had clinically significant pleural effusions. None of the 12 month followup echocardiograms resulted in a serious adverse event. For those subjects completing the 2-year follow-up telephone call, no serious adverse events were reported, and thus, there have been no long term safety events detected at this point.
The cardiac performance measures Resting Total Severity Score (RTSS), percent infarct (“% Infarct”), End Systolic Volume (ESV), and Ejection Fraction (“EF”) were assessed at 3 months post treatment and at 6 months post treatment and compared with controls to assess efficacy of the compositions compared to controls.
SPECT SCAN. As used herein, a single-photon emission computerized tomography (SPECT) scan is a type of nuclear imaging test, which uses a radioactive substance and a special camera to create three dimensional images of the heart to show blood flows to the heart. Generally, the “Resting Total Severity Score (RTSS) is a score based on the amount of technetium not taken up in a SPECT SCAN. The data from Resting Total Severity Score represents cardiac perfusion, i.e., blood flow at the microvascular level, and muscle function. In brief, the technetium used in a SPECT SCAN is taken up by the healthy, perfused heart muscle. Thus, if the heart muscle is healthy and there is adequate blood flow, the muscle will take up the technetium. If the heart muscle is hibernating or apoptotic, technetium uptake is diminished or does not occur at all.
Percent Infarct (MRI). The size of the infarct determines the extent to which a patient will recover from the AMI. A patient who has suffered damage to more than 30 percent of the left ventricle of the heart is twice as likely to die within a year from the injury as a patient who has suffered less damage, and bigger infarcts often require more aggressive therapy. A computer method calculates the amount of damaged tissue by comparing MRI signal strength between damaged and undamaged tissue. Damaged heart tissue is denser than undamaged tissue because the muscle structure has collapsed, and MRI can distinguish between tissues of varying density. The term “percent (%) infarct” as used herein refers to the infarcted area compared to the rest of the heart. For purposes of this study, a % infarct greater than 20% is considered significant.
Change in Myocardial Perfusion and Cardiac Function
Results are shown in Table 25.







TABLE 25





Quantitative Measures of Perfusion and Left Ventricular Function






















Treated
Treated (10
Treated





(5 Million)
Million)
(15 Million)
All Treated


Cardiac Function Test
Control
N = 5)
N = 5)
(N = 6)
(N = 16)
















SPECT

n = 13
n = 5
n = 5
n = 4
n = 14


RTSS


(perfusion)



Baseline
259 +/− 282
714 +/− 657
998 +/− 753
585 +/− 439
778 +/− 619



6 Months
273 +/− 394
722 +/− 520
635 +/− 531
462 +/− 289
616 +/− 448



Difference
14.4 +/− 210 
 7.8 +/− 216
−362 +/− 306  
−122 +/− 260  
−162 +/− 293  



P-value 6
0.80
0.94
0.05
0.41
0.06



month



change


MRI

n = 10
n = 5
n = 4
n = 2
n = 11


LVEF (%)
Baseline
53.2 +/− 10  
46.96 +/− 13  
47.3 +/− 10  
49.9 +/− 7  
47.6 +/− 10  



3 Month
−1.0 +− 5.4  
1.8 +− 6.7
3.4 +/− 5.1
−3.3 +/− 2.7  
1.4 +/− 5.8



Difference



P-value 3-
0.57
0.57
0.26
0.32
0.41



month



change



6 Months
54.2 +/− 11  
46.794 +/− 13   
54 +/− 11
50.1 +/− 6  
50.1 +/− 11  



Difference
1.0 +/− 7.8
−0.02 +/− 13    
6.7 +/− 4  
0.2 +/− 0.8
2.5 +/− 9  



P-value 6
0.68
0.99
0.04
0.78
0.39



month



change


EDV (mL)
Baseline
154.7 +/− 55  
153.3 +/− 30  
176.6 +/− 51  
175.7 +/− 12  
165.8 +/− 36.1 



6 Months
154.1 +/− 55  
176.3 +/− 53  
182.4 +/− 58  
180.1 +/− 41  
179.2 +/− 48  



Difference
−0.56 +/− 20    
23 +/− 37
5.83 +/− 29  
4.39 +/− 29  
13.4 +/− 31  


ESV (mL)
Baseline
76.1 +/− 45  
81 +/− 23
97 +/− 46
88 +/− 18
88 +/− 30



6 Months
74.3 +/− 44  
95 +/− 46
87 +/− 46
91 +/− 32
91.5 +/− 40  



Difference
−1.84 +/− 17    
14 +/− 25
−9.9 +/− 18   
2.69 +/− 13  
3.4 +/− 22 


Infarct Size
Baseline
16.64 +/− 8   
18.8 +/− 8.6 
33.2 +/− 14  
11.7 +/− 1  
22.7 +/− 13  


(% of LV
6 Months
9.95 +/− 9  
16.2 +/− 10.9
22.3 +/− 12  
11.1 +/− 2  
17.5 +/− 11  


Mass)
Difference
−6.7 +/− 5    
−2.6 +/− 5.9  
−10.9 +/− 3    
−0.57 +/− 1    
−5.2 +/− 6    






















P-Value




P-Value
P-Value


(Control + 5




(all
(Control
Combined
Combined
Million




Treated
vs. 10 +
Control
Treated (10
vx. 10 +




vs.
15
(Control +
Million + 15
15



Cardiac Function Test
Control)
Million)
5 Million)
Million)
Million)



















SPECT

n = 13
n = 9
N = 18
n = 9




RTSS



(perfusion)




Baseline


385 +/− 449
814 +/− 635




6 Months


398 +/− 465
558 +/− 425




Difference
0.08
0.02
 13 +/− 205
−255 +/− 297  
0.01




P-value 6


0.79
0.03




month




change



MRI

n = 10
n = 6
n = 15
n =/ −386



LVEF (%)
Baseline


51.1 +/− 11  
48.2 +/− 9  




3 Month


−0.02 +/− 5.8   
1.1 +/− 5.5




Difference




P-value 3-


−.99
0.61




month




change




6 Months


51.8 +/− 12  
52.7 +/− 8  




Difference
0.70
0.33
0.7 +/− 9  
4.5 +/− 5  
0.35




P-value 6


0.77
0.05




month




change



EDV (mL)
Baseline


154.2 +/− 47  
176.4 +/− 39.9 




6 Months


161.5 +/− 53.3 
181.7 +/− 48.7 




Difference
0.24
0.61
 7.3 +/− 28.1
 5.4 +/− 25.9
0.88



ESV (mL)
Baseline


78 +/− 38
94 +/− 37




6 Months


81.3 +/− 44  
88.5 +/− 38  




Difference
0.55
0.66
3.6 +/− 21 
−5.7 +/− 16   
0.34



Infarct Size
Baseline


17.3 +/− 8.2 
26 +/− 16



(% of LV
6 Months


 12 +/− 9.8
18.5 +/− 11  



Mass)
Difference
0.57
0.79
−5.3 +/− 5.8  
−7.5 +/− 5.7  
0.45










Two patients in the control group (refused) and two in the 15 million group (1 death post infusion, 1 acute stent restenosis prior to infusion) did not complete the 6 month SPECT scan. For MRI, 5 patients in the control group (1 death, 2 refused, 2 intracoronary devices (ICD), one in the 10 million group (ICD), and 3 in the 15 million group (1 death, one poor baseline image, 1 ICD) did not complete imaging. Comparison between groups uses data only from those patients completing studies. As shown in Table 25, control patients had the smaller baseline perfusion defect size (RTSS) (p≦0.04) and lower infarct size as a percent of LV mass (p≦0.03) compared to treatment group patients.
As shown in Table 25, the perfusion defect measured as SPECT RTSS remained unchanged at 6 months compared to baseline in controls (259+/−282 to 273+/−394, p=0.80) and patients receiving 5 million CD34+ cells (714+/−657 to 722+/−520, p=0.94). In contrast, patients receiving 10 million and 15 million CD34+ cells experienced a decrease in myocardial perfusion defect (998+/−753 to 635+/−531, p=0.57 and 584+/−439 to 462+/−289, p=0.41, respectively). The decrease in perfusion defect was significant (−255+/−297, p=0.03) and significantly greater among patients receiving >10 million CD34+ when compared to controls (p=0.02) and the controls and 5 million group combined (p=0.01). Of note, 9 of 10 patients (90%) receiving >10 million CD34+ cells had a drop in RTSS compared as compared to only 7 of 13 controls (54%) and 2 of 5 patients receiving 5 million cells (40%). The one patient in the 15 million group whose RTSS did not drop was the only treated patient (all groups) whose CD34+ SDF-1 mobility was 1.0% (0.43%).
There was a trend to a greater reduction in the myocardial perfusion defect in the treated group compared to the controls, p=0.08. A post-hoc analysis examined changes in myocardial perfusion among subjects receiving the two higher doses (10 million or 15 million cells) versus the low dose (5 million cells) or control therapy. Subjects receiving ≧10 million CD34+ cells had a significant improvement in RTSS compared to those receiving 5 million cells or no cell therapy, p=0.02. Results were not influenced by demographic characteristics, baseline ventricular dysfunction or baseline infarct size.
As shown in Table 25, there were no significant differences in the change in these parameters in the treated compared to the control groups after 6 months. Because improvement in perfusion was observed only in the two higher dose levels, a post-hoc analysis examining changes in cardiac function among subjects receiving high dose CD34+ cell infusion (10 million or 15 million cells) versus low dose infusion (5 million cells) and controls. Whereas there was no change in LVEF (+0.7%, p=0.68) in controls and the 5 million cell group, there was a strong trend towards improvement in LVEF (+4.5%, p=0.059) in those receiving ≧10 million CD34+ cells. Although similar trends were observed with changes in end-systolic volume, end diastolic volume, and infarct size reduction, these changes did not reach statistical significance.
CD34+ Cell Mobility in an SDF-1 Gradient
To investigate whether either CXCR-4 expression or SDF-1 mobility of the infused CD34+ cells influenced outcomes, the relationship between the number of infused cells expressing CXCR-4 and SDR-1 mobility and the changes in RTSS and infarct size was assessed. The results are shown in FIG. 4.
FIG. 4(A) shows the change in infarct size as a percent of LV mass versus CD34+ dose times % of CD34+ cells mobile in an SDF gradient. FIG. 4(B) shows the change in perfusion defect (RTSS) versus CD34+ dose time % of CD34+ cells mobile in an SDF gradient. In the entire treated cohort, there was a significant correlation between the quantity of CD34+/SDF-1 mobile cells infused (product of CD34+ cells and SDF-1 mobility) and both the decrease in RTSS (r2=0.45, p=0.011) and infarct size as a percent of LV mass (r2=0.49, p=0.015), suggesting that greater SDF-1 mobility was associated with greater reduction in infarct size and improved infarct region perfusion. However, there were no significant correlations between the number of CD34+/CSCR-4+ cells infused, CD34+/VEGF+ cell infused, or colony forming (CFU) ability and change in RTSS or infarct size, likely because there was no correlation between CSCR-4 expression and SDF-1 mobility. No significant correlations were identified between changes in LVEF or LVESV and cell characteristics (data not shown).
Discussion
These results show that IRA infusion of ≧10 million CD34+ cells was safe and associated with a significant (31%) reduction in RTSS at 6 months compared to baseline. In contrast, patients receiving 5 million CD34+ cells and controls had no significant change in RTSS. RTSS is a composite of the extent and severity of the perfusion defect assessed b SPECT, and potentially is also an index of cardiomyocyte viability that has been validate to detect 10% or greater differences in single individuals on repeat measures. Further supporting a dose threshold effect, at 6 months compared to baseline, there was a trend in improvement in LVEF among patients receiving ≧10 million CD34+ cells when compared to controls, patients receiving 5 million CD34+ cells and the combined control and 5 million groups. Larger infarct size after stenting, particularly if associated with a more extensive perfusion defect, results in a greater likelihood of adverse ventricular remodeling manifesting in a decrease in LVEF and increase in LVESV over time. In the study described, at baseline, patients receiving ≧10 million CD34+ cells had larger infarct sizes and greater RTSS. However, despite this, there was a greater reduction in RTSS and improvement (trend) in LVEF in these patients. In addition, among patients in the combined 5 million and control groups, mean LVEF did not improve at 3 months or 6 months, and LVESV steadily increased from baseline, consistent with adverse ventricular remodeling. Strikingly, among patients receiving ≧10 million CD34+ cells, despite beginning with greater infarcts and perfusion defects, LVEF improved at 3 months and to a greater extent at 6 months, while LVESV improved from 3 months to 6 months after worsening during the first three months, collectively suggesting an ongoing therapeutic effect.
As shown in Table 26, in the 10 and 15 million cohort, none of the patients had a drop in LVEF greater than 1% (ie none had a clinically relevant drop in LVEF) where as 30% of controls and 40% of the 5 million group did (range −2.9 to −17.4%). This is consistent with the conclusion that the chemotactic hematopoietic stem cell product of the invention can prevent ventricular remodeling (clinically manifested in a drop in LVEF at 6 months).







TABLE 26







6-Month Change in RTSS and LVEF.










RTSS
LVEF


Patient
6 Month Change
6 Month Change












Cohort 1




1
−71
18.2%


2
164
5.2%


3
46
1.6%


4
−322
−12.7%


5
222
−12.4%


Cohort 2


1
−859


2
−97
7.7%


3
−427
6.0%


4
−294
11.4%


5
−137
1.8%


Cohort 3


1


2
263
0.8%


3
−274


4
−287


5
−190
−0.4%


Control


1
−210
−2.98%


2
−99
4.60%


3
342
2.12%


4
0


5
−250
3.24%


6
528
5.90%


7
−54


8
13
4.60%


9
−30
−17.43%


10
1


11
−584
−3.70%


12
78
11.20%


13
−93
2.90%


14
−38


15
−1266









In addition to the number of cells infused, the described data indicates that dose threshold for patients is dependent on the number and mobility of the cells infused, and that mobility declines over time with a median 57% drop between 24 and 48 hours after completion of marrow harvest and a further 11% by 72 hours. In fact, among patients receiving ≧10 million CD34+ cells, the one patient who did not experience a decrease in RTSS had the lowest CD34+ cell mobility of all patients tested (0.43%).
Example 12
Multiple Administrations of Chemotactic Hematopoietic Stem Cell Product to Subjects
The blood supply in the peri-infarct ischemic border zones, is marginal, placing the cardiomyocytes of the border zone in jeopardy. Multiple administrations of chemotactic hematopoietic stem cell product, by supporting cells in the border zone, may preserve/restore viability of the periinfarct myocardium.
According to this aspect of the described invention, a first aliquot of the composition is administered at a first infusion date, a second aliquot of the composition is administered at a second infusion date, and a third aliquot of the composition is administered at a third infusion date. The scheduling of the second and third infusion dates is determined for a given patient by the treating practitioner according to his/her medical judgment. According to one embodiment, the composition is administered at a first infusion date, 30 days after the first infusion date at a second infusion date, and 60 days after the first infusion date at a third infusion date.
Eligible subjects/patients presenting with symptoms and clinical findings suggestive of a myocardial infarction and eligible for inclusion in the study will be selected as described in Example 1 and catheterized as described in Example 2. The bone marrow comprising potent CD34+ cells will be acquired from the subject/patient as described in Example 3 and, in some embodiments, the harvested bone marrow will be transported to the processing facility as described in Example 4. CD34+ cells will be selected from the harvested bone marrow product as described in Example 5.
The Isolex 300i system will be used to process the RBC-depleted product or the bone marrow product whose RBC volume is <20 ml according to the following processing steps:
(i) The bone marrow is washed automatically to remove platelets;
(ii) CD34 positive (CD34+) cells are labeled specifically for selection by incubation with the Isolex 300i CD34 monoclonal antibody (Mab);
(iii) Unbound reagent is removed by washing the cell suspension with buffer solution;
(iv) Sensitized CD34+ cells (meaning CD34+ cells labeled with CD34 Mab) are captured by Dynabeads M-450 Sheep anti-Mouse IgG;
(v) A selection column is used to separate the magnetically-labeled Dynabeads having captured CD34+ cells from unwanted cells, which are washed through the selection column and collected in the Negative Fraction Bag; and
(vi) PR34+Stem Cell Releasing Agent releases CD34+ cells from the column, and the CD34+ cells are collected in the End Product Bag. The system performs several washing steps, disposing of most of the liquid into the Buffer Waste Bag.
The Isolex® selected CD34+ fraction then will be assayed to determine WBC and CD34+ cell yields as described in Example 6. A first aliquot of the chemotactic hematopoietic stem cell product containing at least 10×106 CD34+ cells will be formulated a described in Example 7, transported to the catheterization facility as described in Example 8, and infused into the patient as described in Example 9 at the first infusion date. At least two additional aliquots of the chemotactic hematopoietic stem cell product containing at least 10×106 CD34+ cells per aliquot will be frozen at '70 C for subsequent administration. (see “Cryopreservation Study” below).
CryoPreservation Study.
This study was conducted to evaluate the ability of the Isolex-based portion of the chemotactic hematopoietic stem cell product manufacturing process to effectively enrich for CD34+ cells of the cryopreserved MMH. The protocol has been designed to evaluate the yield, viability, functionality and stability of the CD34+ cells derived from the enrichment of cryopreserved MMH. The study has been designed to evaluate and describe the effect on the chemotactic hematopoietic stem cell product of cryopreservation of the RBC reduced MMH prior to the Isolex-based CD34 selection.
The following experimental conditions have been applied: (1) Two (2) MMH for each of three (3) replicates in order to provide for adequate cell yield to meet with requirements of the experimental design; with a twenty four (24) hour interval between MMH and commencement of RBC depletion procedure.
(2) Study control: Freshly prepared chemotactic hematopoietic stem cell product, with full product characterization after perfusion of the chemotactic hematopoietic stem cell product through a catheter at 48 and 72 hours after MMH.
(3) Experimental: the chemotactic hematopoietic stem cell product derived from cryopreserved MMH, with full product characterization after perfusion of the chemotactic hematopoietic stem cell product derived from cryopreserved MMH through a catheter at 48 and 72 hours after MMH, minus the time the cryopreserved MMH remains in storage (defined as ≧24 hours)
Study Design
In order to yield sufficient CD34+ cells to perform the intended experiment, two (2) donors will be required. More than or equal to 80 ml MMH and ≧30 ml of peripheral blood will be collected from each donor.
In-bound storage: Samples will be stored at 2 to 8° C. for twenty four (24) hour before commencing the RBC reduction procedure.
Following RBC reduction, the MMH from both donors will be pooled and then divided into two equal fractions. One fraction will be used as a fresh (unfrozen) product control and the other fraction will be used for the cryopreservation test.
For the cryopreservation test, RBC reduced MMH will be frozen in a −86° C. freezer and then cryostored in the vapor phase (≦−150° C.) of a liquid nitrogen freezer (LNF) using the cryoprotectant containing the liquid source Hetastarch (6% Hetastarch in 0.9% Sodium Chloride manufactured by Hospira). The procedure for preparation of the cryoprotectant and freezing and cryostorage of the RBC reduced MMH is as follows. 145 ml of 25% HSA (Human Serum Albumin, Baxter, 060-998 or equivalent) and 71.4 mL of DMSO (Cryoserv) are transferred into one (1) bag of 500 ml 6% Hetastarch (Hospira, 0074-7248-03 or equivalent). The complete cryoprotectant contains 10% DMSO, 4.2% Hetastarch, and 5% HSA). The Hetastarch bag is inverted about 10 times to mix. The prepared cryprotectant is stored in a 2-8 C refrigerator for at least 2 hours and not more than 24 hours after preparation. The required volume of cryoprotectant is transferred into the MMH preparation so that the final concentrations of DMSO, Hetastarch and HSA in the human progenitor cell product are 5%, 2.1%, and 2.5% respectively. Samples of the MMH product with cryoprotectant added is collected for Trypan Blue Exclusion analysis (viability), WBC count, and sterility. The tubing of the prepared freezing bag is heat-scaled, the MMH product volume for each prepared freezing bag is determined, and the freezing bag is placed inside a freezing canister/cassette. The freezing canister/cassette is placed horizontally inside a −86 C mechanical freezer. The MMH product may be stored in the −86 C mechanical freezer for short-term storage up to six (6) months. For long-term storage, the cassette(s) are removed from the −86 C mechanical freezer and placed into liquid nitrogen freezer (LNF) for storage in the vapor phase of liquid nitrogen along with the sample(s) of the MMH product for analysis.
Both control (unfrozen) and cryopreserved (after thaw) samples will be Isolex processed essentially as described in Example 5 above. Samples in two 10 ml syringes will be prepared from the selected CD34+ cells. Full product characterization will be performed at the following time points: (i) After perfusion of the product through a catheter at 48 hours after MMH; and (ii) after perfusion of the product through a catheter at 72 hours after MMH. For the cryopreserved samples, the term “72 hours of collection”, for example, means the time from collection to the time of testing, excluding the time elapsed from freezing and cryostorage of the RBC depleted bone marrow.
Key determinants for the CD34+ cell quality of the hematopoietic stem cell product include: (i0 CD34+ cell enumeration and 7-AAD viability; (ii) SDF-1/CXCR-4 mediated CD34+ cell migratory activity; (iii) expression of CXCR-4 cell surface antigen on CD34+ cells; and (iv) growth of hematopoietic progenitor cell colonies (CFU). This experiment will be repeated three times.
Summary of Results
The study was conducted in accordance with the methods described above. All deviations from methodology and materials used are detailed in the related result sections presented below.
Table 27 summarizes the relevant information on the donors of the bone marrow used in this study.







TABLE 27







Age and gender of the bone marrow donors for the


cryopreservation study.














Exp 1

Exp 2

Exp 3
















Donor
1
2
3
4
5
6







Age
26
26
22
62
32
24



Gender
F
F
F
F
F
F










Table 28 summarizes the sample volume, RBC content and the yield, viability and purity of the cells in the pre-processed MMH following 24-h storage in a 2-8° C. refrigerator.







TABLE 28







Post 24 hours storage at 2-8° C. - Volume, cell yield and quality of MMH.











Exp 1
Exp 2
Exp 3














Donor 1
Donor 2
Donor 3
Donor 4
Donor 5
Donor 6

















Volume (ml)
117
64
106
105
103
113


WBC per μl#
1.39E+04
1.26E+04
1.39E+04
1.44E+04
1.94E+04
2.45E+04


TNC#
1.62E+09
8.03E+08
1.47E+09
1.51E+09
1.99E+09
2.76E+09


HCT#
33.85%
33.40%
29.10%
27.85%
31.60%
32.60%


RBC vol. (ml)#
39.44
21.38
30.85
29.24
32.55
36.84


CD45+ cell viability*
91.13%
91.72%
90.58%
93.17%
94.11%
95.8%


Viable CD34+ cell
149.18
148.38
140.89
114.45
150.80
203.76


per μl*


CD34+ cell viability*
94.14%
98.90%
98.35%
97.24%
98.89%
98.78%


CD34+ cell purity*
1.44%
1.32%
1.23%
0.97%
1.21%
0.88%


CXCR4 expressing
77.68%
77.03%
71.88%
64.57%
75.75%
68.36%


CD34+ cells~


Total # of CD34+
1.74E+07
9.50E+06
1.49E+07
1.20E+07
1.55E+07
2.30E+07


cells*





#Determined by hematology analyzer


*Determined by flow cytometric analysis of CD45-FITC/CD34-PE antibodies and 7-AAD staining of the sample


~Determined by flow cytometric analysis of CD34-FITC and CXCR4-PE antibodies staining of the sample






In each of the experiments, the MMH from each pair of donors were pooled following RBC reduction.
Table 29 presents the RBC content, viability and cell recovery of pooled MMH after RBC reduction:







TABLE 29







Post RBC reduction - RBC content and cell quality











Exp 1
Exp 2
Exp 3



Donors 1 & 2
Donors 3 & 4
Donors 5 & 6














RBC volume
15.35 ml
13.80 ml
20.85 ml


TNC recovery#
76.95%
85.93%
89.37%


CD45+ cell viability
84.97%
93.35%
95.60%


CD34 recovery#
72.89%
84.00%
88.36%


CD34+ cell viability
93.99%
97.92%
98.95%


CXCR4 expressing
71.33%
64.89%
74.64%


CD34+ cells





#As compared to the pre-processed samples






Following RBC reduction, each of the pooled MMH samples was divided into two equal fractions. One was used as a fresh (unfrozen) control and the other one was used for the cryopreservation test.
For cryopreservation, MMH mixed with an equal volume of chilled cryoprotectant was loaded evenly into two 250 ml Cryocyte containers, frozen in a mechanical freezer (−86° C.) and then stored cryopreserved in the vapor phase of a LNF according to the Protocol. Table 30 presents data obtained from post-thawed and washed MMH:







TABLE 30







Post thawed & washed MMH - Cell quality and recovery of cells


Cryopreserved Sample Thawed and washed











Exp 1
Exp 2
Exp 3














Storage duration prior
10 days
8 days
8 days


to thaw


Wash media
PBS working
2% Dextran~
8.3% Dextran@



sol'n*


RBC volume
0.39 ml
1.11 ml
0.38 ml


TNC recovery#
36.11%
50.73%
28.61%


CD45+ cell viability
61.85%
32.18%
43.97%


CD34+ cell recovery#
52.43%
46.29%
15.72%


CD34+ cell viability
94.36%
86.11%
81.76%


CD34+ cell purity
2.40%
1.29%
1.88%


CXCR4 expressing CD34+
51.42%
50.74%
37.85%


cells





#As compared to the RBC reduced MMH before cryopreservation.


*PBS Working Solution contained 1% HSA and 0.41% sodium citrate (w/v) in PBS (Ca++ and Mg++ free). Washing of cells with this solution was performed according to that instructed in the Protocol.


~This wash solution contained 2% Dextran 40, 1% HSA and 0.4% Na citrate in PBS (Ca++ and Mg++ free). The thawed sample was expanded with 200 ml of this solution and was then washed twice each with 200 ml of this solution. Centrifugation was set for 600 g, 10 minutes at 20° C. The washed cells were resuspended with 150 ml PBS Working Solution for Isolex process.


@This solution contained 8.3% Dextran 40 and 4.2% HSA in saline. The washing procedure was essentially as described for the 2% Dextran 40 wash solution.






Table 31 summarizes the CD34+ cell quality and recovery of the chemotactic hematopoietic stem cell product1 prepared from the unfrozen and cryopreserved MMH following Isolex processing.







TABLE 31







Post Isolex - Cell quality and recovery of cells











Exp 1
Exp 2
Exp 3













MMH source
Unfrozen
Frozen
Unfrozen
Frozen
Unfrozen
Frozen





CD34+ cell
47.28%
37.88%
35.94%
49.29%
44.05%
82.25%


recovery#


CD34+ cell viability#
99.37%
96.89%
98.97%
95.05%
98.26%
95.38%


CD34+ cell purity
87.51%
83.95%
86.47%
81.91%
81.71%
50.87%


Total # of viable
4.63E+06
1.95E+06
4.07E+06
2.58E+06
7.50E+06
2.20E+06


CD34+ cells





#As compared to the RBC reduced sample for unfrozen samples and post thawed and washed samples for frozen samples.






Following Isolex processing of each RBC reduced MMH pooled pair, two chemotactic hematopoietic stem cell product (“AMR-001”) samples with equal number of CD34+ cells, each in a 10 ml syringe, were prepared. Both AMR-001 samples were stored at 2-8° C. for stability testing. At 48 and 72 hours from MMH (For cryopreserved MMH samples, the time for cryostorage was not included), a prepared AMR-001 was perfused through a balloon dilatation catheter performed in a manner as for a clinical AMR-001. A full CD34+ cell characterization was performed on the perfused AMR-001 samples and the results are presented in Tables 32, 33, 34, and 35. Table 36 shows the balloon dilatation catheter used.







TABLE 32







Post infusion through catheter - CD34+ cell purity, viability and recovery









Catheter perfused AMR-001









MMH source










Unfrozen
Frozen









Time post MMH












Experiment

48 h
72 h
48 h
72 h















1
CD34+ cell recovery#
101.73%
92.32%
91.71%
69.35%



CD34+ cell viability
99.08%
98.13%
94.98%
91.80%



CD34+ cell purity
85.92%
84.93%
82.94%
74.24%



Total # of CD34+ cells
2.36E+06
2.14E+06
8.92E+05
6.74E+05


2
CD34+ cell recovery#
95.65%
89.20%
77.10%
74.01%



CD34+ cell viability
98.29%
97.29%
89.47%
82.82%



CD34+ cell purity
81.49%
82.42%
75.30%
70.50%



Total # of CD34+ cells
1.95E+06
1.81E+06
9.96E+05
9.56E+05


3
CD34+ cell recovery#
104.17%
101.99%
77.35%
79.12%



CD34+ cell viability
98.46%
97.51%
86.86%
85.59%



CD34+ cell purity
83.18%
82.80%
47.81%
43.71%



Total # of CD34+ cells
3.91E+06
3.83E+06
8.52E+05
8.71E+05





#As compared with the prepared AMR-001 before perfusion













TABLE 33







Post infusion through catheter - CXCR4 expressing CD34+ cells (% of


total CD34+ cells).









MMH source of AMR-001 samples











Exp 1
Exp 2
Exp 3













Catheter perfusion
Unfrozen
Frozen
Unfrozen
Frozen
Unfrozen
Frozen





48 h post MMH
66.52%
53.31%
57.64%
41.35%
60.14%
54.16%


72 h post MMH
73.87%
53.89%
56.73%
44.07%
64.60%
50.67%
















TABLE 34







Post infusion through catheter - Migratory CD34+ cells (% of total viable


CD34+ cells).









MMH source of AMR-001 samples










Catheter
Exp 1
Exp 2
Exp 3













perfusion
Unfrozen
Frozen
Unfrozen
Frozen
Unfrozen
Frozen





48 h post
18.81 ± 1.83%*
5.87 ± 1.98%
19.67 ± 10.43%
15.67 ± 2.24%
24.89 ± 1.93%
26.66 ± 1.53%


MMH


72 h post
(1.07%)#
(1.51%)
(1.06%)
(2.19%)
(1.44%)
(1.56%)


MMH





*SDF-1 induced migration. % of migratory CD34+ cell of total viable CD34+ cells with standard deviation of three replicates.


#Natural migration (no SDF-1 added)













TABLE 35







Post infusion through catheter - Number of CFU per 100 viable CD34+


cells cultured.









MMH source of AMR-001 samples











Exp 1
Exp 2
Exp 3













perfusion
Unfrozen
Frozen
Unfrozen
Frozen
Unfrozen
Frozen
















48 h post MMH
24
15.5
31.5
14
38
15.5


72 h post MMH
20.5
0.05
62.5
12
30.5
7
















TABLE 36







Balloon dilatation catheters used















MMH









source
Time of



of the
perfusion



AMR-
(Hours



001
of

Balloon


Exp
sample
MMH)
Manufacture
length/dia.
Catalog #
Lot #
Comment

















1
Unfrozen
48 h
Sprinter
12/3.5 mm
SPR3512W
258795
Outdated




72 h
Sprinter
12/4.0 mm
SPR4012W
254243
Outdated



Frozen
48 h
Sprinter
15/3.0 mm
SPR3015W
412090
Outdated




72 h
Voyager
15/3.0 mm
1009443-
8111462
—







15


2
Unfrozen
48 h
Sprinter
15/3.5 mm
SPR3515W
443152
Outdated




72 h
Sprinter
15/3.5 mm
SPR3515W
443152
Outdated



Frozen
48 h
Voyager
15/3.0 mm
1009443-
8111462
—







15




72 h
Voyager
15/3.0 mm
1009443-
8092561
—







15


3
Unfrozen
48 h
Voyager
15/3.0 mm
1009443-
8111462
Reused*







15




72 h
Sprinter
15/3.0 mm
SPR3015W
476734
Outdated



Frozen
48 h
Sprinter
15/3.0 mm
SPR3015W
476734
Outdated




72 h
Sprinter
15/3.0 mm
SPR3015W
476734
Outdated





*Prior to be used for the 2nd time, the catheter and the central lumen were 1st washed and flushed with 70% isopropyl alcohol and then with sterile PBS. The central lumen was then injected with air in order to remove the residual liquid inside. The washing procedure was performed inside a bio-safety cabinet.






Discussion
The aim of this study was to evaluate the quality of AMR-001 manufactured from cryopreserved MMH.
Post Isolex CD34+ cell recovery of the AMR-001 manufactured from unfrozen MMH (Control samples) was on average 34.6±4.35% (range 303% to 39%) which is within the acceptance range for manufacture of AMR-001 for clinical use. It should be noted that the data presented above are estimated without taking account for the cells removed for the in-process tests, therefore the actual CD34+ cell recovery will be slightly higher than that presented.
Post catheter CD34+ cell recovery was 100.52±4.39% (95.65% to 104.17%) at 48 hours post MMH and 94.50±6.67% (89.20% to 101.99%) at 72 hours post MMH. There was no substantial reduction in viability (Table 32), CXCR-4 expression (Table 33), migratory activity (Table 34) and CFU growth (Table 35) of CD34+ cells at 72 hours post MMH as compared to those monitored at 48 hours post MMH.
For the cryopreservation test, RBC reduced MMH samples were cryopreserved according to the protocol for cryopreservation of bone marrow for transplantation where MMH samples mixed with equal volume of cryoprotectant with final concentration of 5% DMSO, 2.5% HSA and 2.1% Hetastarch (from liquid source 6% Hetastarch, Hospira) were frozen at −86° C. and then cryostored in the vapor phase of a liquid nitrogen freezer (LNF).
Post cryopreservation and thaw, the stability, viability, mobility and growth in culture of Isolex selected CD34+ cells is maintained. Thus, the frozen-thawed cells meet the criteria for clinical use.
In some embodiments, a second aliquot comprises the frozen chemotactic hematopoietic stem cell product product. In some embodiments, the aliquot will be thawed 30 days after the first infusion date, and samples of the thawed chemotactic hematopoietic stem cell product will be removed to be assayed for WBC count, by flow cytometry (for CD34+ cell enumeration and viability), Gram stain, and sterility. The thawed chemotactic hematopoietic stem cell product will be released for infusion 1 to 2 days after thawing only if it meets the following criteria:

    
    
        CD34+ cell purity of at least about 70%, 75%, 80%, 85%, 90% or 95%;
        A negative Gram stain result for the selected positive fraction;
        Endotoxin Levels: less than about 0.5 endotoxin units/ml;
        Viable CD34+ cell yield of the “Chemotactic hematopoietic stem cell product” meets the required dosing as per the treatment cohort;
        CD34+ cells are at least about 70%, 75%, 80%, 85%, 90% or 95% viable by 7-AAD;
        USP sterility result for “Positive Fraction Supernatant”: negative (14 days later).
    
    


Sterility assessment on the stem cell product including gram staining and endotoxin will be performed prior to product release for infusion. USP sterility (bacterial and fungal) culture will be performed and the results will be reported to the principal investigator. In the event of a positive USP sterility result, the subject and attending physician on call will be notified immediately, provided with identification and sensitivity of the organism when available, and documentation of appropriate anti-microbial treatment and treatment outcome will be recorded by the investigative site and the sponsor.
The second aliquot comprising the thawed chemotactic hematopoietic stem cell product will be formulated as described in Example 7, transported to the catheterization facility as described in Example 8, and infused into the patient as described in Example 9 at the second infusion date. In some embodiments, the second infusion date is 30 days after the first infusion date.
In some embodiments, a third aliquot comprising the frozen chemotactic hematopoietic stem cell product product will be thawed 60 days after the first infusion date, and samples of the third aliquot comprising the thawed chemotactic hematopoietic stem cell product will be removed to be assayed for WBC count, by flow cytometry (for CD34+ cell enumeration and viability), Gram stain, and sterility. The thawed chemotactic hematopoietic stem cell product of the described invention will be released for infusion 1 to 2 days after thawing only if it meets the following criteria:

    
    
        CD34+ cell purity of at least about 70%, 75%, 80%, 85%, 90% or 95%;
        A negative Gram stain result for the selected positive fraction;
        Endotoxin Levels: less than about 0.5 endotoxin units/ml;
        Viable CD34+ cell yield of the “Chemotactic hematopoietic stem cell product” meets the required dosing as per the treatment cohort;
        CD34+ cells are at least about 70%, 75%, 80%, 85%, 90% or 95% viable by 7-AAD; and
        USP sterility result for “Positive Fraction Supernatant”: negative (14 days later)
    
    


Sterility assessment on the stem cell product including gram staining and endotoxin will be performed prior to product release for infusion. USP sterility (bacterial and fungal) culture will be performed and the results will be reported to the principal investigator. In the event of a positive USP sterility result, the subject and attending physician on call will be notified immediately, provided with identification and sensitivity of the organism when available, and documentation of appropriate anti-microbial treatment and treatment outcome will be recorded by the investigative site and the sponsor.
The thawed chemotactic hematopoietic stem cell product of the third aliquot will be formulated as described in Example 7, transported to the catheterization facility as described in Example 8, and infused into the patient as described in Example 9 at the third infusion date. In some embodiments, the third infusion date will be 60 days from the first infusion date.
It is proposed that administration of a potent cell dose timed properly post-AMI according to the described invention may result in a reduction in major adverse cardiac events, including, but not limited to, premature death, recurrent myocardial infarction, congestive heart failure, significant arrythmias, and acute coronary syndrome.
Example 13
Co-Administration of the Chemotactic Hematopoietic Stem Cell Product with an Agent that can Promote Cardiomyocyte Growth
The described invention demonstrates the prevention of cardiomyocyte loss after AMI through enhancement of perfusion and prevention of apoptosis. Further restoration of cardiac function requires significantly increasing the regenerative capacity of cardiomyocytes. Regenerating cardiomyocytes will require adequate perfusion to allow for sustainable cardiomyocyte growth, or will suffer the consequences of ischemia including hibernation and apoptosis.
It is proposed that a pharmaceutical composition comprising the chemotactic hematopoietic stem cell product of the present invention and at least one compatible active agent that promotes cardiomyocyte growth, can (i) restore cardiac function after AMI and (ii) prevent major adverse cardiac events. In one embodiment, the pharmaceutical composition comprising the chemotactic hematopoietic stem cell product of the present invention and at least one compatible active agent that promotes cardiomyocyte growth increases perfusion in the peri-infarct zone. In another embodiment, the pharmaceutical composition comprising the chemotactic hematopoietic stem cell product of the present invention and at least one compatible active agent that promotes cardiomyocyte growth increases perfusion in hibernating myocardium. In another embodiment, the pharmaceutical composition comprising the chemotactic hematopoietic stem cell product of the present invention and at least one compatible active agent that promotes cardiomyocyte growth rescues cardiomyocytes from apoptosis. In another embodiment, the pharmaceutical composition comprising the chemotactic hematopoietic stem cell product of the present invention and at least one compatible active agent that promotes cardiomyocyte growth rescues cardiomyocytes from hibernation. In another embodiment, the pharmaceutical composition comprising the chemotactic hematopoietic stem cell product of the present invention and at least one compatible active agent that promotes cardiomyocyte growth generates new cardiomyocytes to replace lost cardiomyocytes. In another embodiment, the pharmaceutical composition comprising the chemotactic hematopoietic stem cell product of the present invention and at least one compatible active agent that promotes cardiomyocyte growth prevents left ventricular remodeling.
In one embodiment, the at least one compatible active agent that promotes cardiomyocyte growth comprises neuregulin 1. Recombinant human neuregulin 1 will be obtained from commercial sources. (Cell Sciences, Novus Biologicals, R & D Systems, Raybiotech, Inc., Shenandoah Biotechnology, Spring Bioscience). Additional compatible active agents that promote cardiomyocyte growth include, but are not limited to, vascular endothelial growth factor (VEGF)-A, VEGF-B, VEGF-C, VEGF-D, placental growth factor (PIGF), catecholamines, such as, but not limited to, norepinephrine (Adams, J. W., and Brown, J. H. Oncogene. 20(14):1626-1634, 2001; Laks, M. M., et al. Chest. 64:75-78, 1973), endothelin-1 (Adams, J. W., and Brown, J. H. Oncogene. 20(14):1626-1634), a prostaglandin Fα (Adams, J. W., and Brown, J. H. Oncogene. 20(14):1626-1634), angiotensin II (Adams, J. W., and Brown, J. H. Oncogene. 20(14):1626-1634), phorbol esters (Schluter, K. D., and Piper, H. M. FASEB J. 13:S17-S22, 1999), neuropeptide Y (Schluter, K. D., and Piper, H. M. FASEB J. 13:S17-S22, 1999), transforming growth factor β1 (TGF-1β), Gq protein (Schluter, K. D., and Piper, H. M. FASEB J. 13:S17-S22, 1999; Adams, J. W., and Brown, J. H. Oncogene. 20(14):1626-1634), diacyl glyercol (DAG) (Schluter, K. D., and Piper, H. M. FASEB J. 13:S17-S22, 1999), salusin-α (Yu, F., et al. Regul. Pep. 122(3):191-197, 2004), salusin-β (Yu, F., et al. Regul. Pep. 122(3):191-197, 2004), insulin-like growth factor (IGF-1) (Davis, M. E., et al. Proc. Natl. Acad. Sci. USA. 103(21):8155-8160, 2006), myostatin (Sharma, M., et al. J. Cell. Physiol, 180(1):1-9, 1999), granulocyte colony-stimulating factor (G-CSF) (Ohki, Y., et al. FASEB J. doi:10.1096/fj.04-3496fje), macrophage colony-stimulating factor (M-CSF) (Okazaki, T., et al. Am. J. Pathol. 171:1093-1103, 2007), TWEAK, thiazolidinediones, such as, but not limited to, rosiglitazone (Duan, S. Z., et al. Circul. Res. 97:372-379, 2005), and variants or recombinant derivatives thereof. Each of the cited references is incorporated by reference in its entirety herein.
Increasing doses of at least one of neuregulin 1, vascular endothelial growth factor (VEGF)-A, VEGF-B, VEGF-C, VEGF-D, placental growth factor (PIGF), catecholamines, such as, but not limited to, norepinephrine, endothelin-1, prostaglandin F2α, angiotensin II, phorbol esters, neuropeptide Y, active transforming growth factor β1 (TGF-1β), Gq protein, diacyl glyercol (DAG), salusin-α, salusin-β, insulin-like growth factor (IGF-1), myostatin, granulocyte colony-stimulating factor (G-CSF), macrophage colony-stimulating factor (M-CSF), TWEAK, thiazolidinediones, such as, but not limited to, rosiglitazone, and variants or recombinant derivatives thereof, will be admixed with the chemotactic hematopoietic stem cell product of the described invention and tested in vitro after passage through a catheter for product viability, sterility, purity and potency, meaning viability, migratory capacity and CFU-growth, after storage for up to 72 hours. If potency, purity and viability are maintained, purified, sterile human derived CD34+ cells containing a subpopulation of potent cells expressing CXCR-4 and having CXCR-4-mediated chemotactic activity will be infused via the tail vein in Nod SCID mice after coronary artery ligation and relief (induced AMI model). The effect of this treatment on cardiac perfusion, cardiac muscle function, histopathology, apoptosis, and scarring will be assessed post infusion and compared to controls (i.e., Nod SCID mice not receiving cells). Prior studies have demonstrated an improvement in perfusion, human neoangiogenesis, prevention of apoptosis, and preserved cardiac function in treated versus control animals. Next, increasing doses of at least one of neuregulin 1, vascular endothelial growth factor (VEGF)-A, VEGF-B, VEGF-C, VEGF-D, placental growth factor (PIGF), catecholamines, such as, but not limited to, norepinephrine, endothelin-1, prostaglandin F2α, angiotensin II, phorbol esters, neuropeptide Y, active transforming growth factor β1 (TGF-1β), Gq protein, diacyl glyercol (DAG), salusin-α, salusin-β, insulin-like growth factor (IGF-1), myostatin, granulocyte colony-stimulating factor (G-CSF), macrophage colony-stimulating factor (M-CSF), TWEAK, thiazolidinediones, such as, but not limited to, rosiglitazone, and variants or recombinant derivatives thereof, will be added to the purified, sterile human derived CD34+ cells containing a subpopulation of potent cells expressing CXCR-4 and having CXCR-4-mediated chemotactic activity of the described invention and the results will be compared to control animals and to animals treated with the purified, sterile human derived CD34+ cells containing a subpopulation of potent cells expressing CXCR-4 and having CXCR-4-mediated chemotactic activity of the described invention alone.
If such a pre-clinical model shows a synergistic beneficial effect with the purified, sterile human derived CD34+ cells containing a subpopulation of potent cells expressing CXCR-4 and having CXCR-4-mediated chemotactic activity of the described invention combined with the at least one compatible active agent that promotes cardiomyocyte growth, a dose escalation safety and efficacy trial in sustaining and in AMI patients is proposed. For this study, patients will receive the purified, sterile human derived CD34+ cells containing a subpopulation of potent cells expressing CXCR-4 and having CXCR-4-mediated chemotactic activity of the invention with or without the at least one compatible active agent that promotes cardiomyocyte growth. The at least one compatible active agent that promotes cardiomyocyte growth will be administered in increasing doses to determine (i) the mean therapeutic dose (MTD) and (ii) whether perfusion and cardiac function are enhanced by the combination of neuregulin 1 and the purified, sterile human derived CD34+ cells containing a subpopulation of potent cells expressing CXCR-4 and having CXCR-4-mediated chemotactic activity of the described invention compared to the purified, sterile human derived CD34+ cells containing a subpopulation of potent cells expressing CXCR-4 and having CXCR-4-mediated chemotactic activity of the described invention alone.
While the described invention has been described with reference to the specific embodiments thereof, it should be understood by those skilled in the art that various changes may be made and equivalents may be substituted without departing from the true spirit and scope of the invention. In addition, many modifications may be made to adapt a particular situation, material, composition of matter, process, process step or steps, to the objective, spirit and scope of the described invention. All such modifications are intended to be within the scope of the claims appended hereto.

## Claims

1. A method for treating adverse ventricular remodeling due to an infarct area injury resulting from acute myocardial infarction, comprising
(a) acquiring from a subject that has suffered an acute myocardial infarction an isolated population of autologous mononuclear cells comprising CD34+ cells;
(b) sterilely enriching the isolated population of autologous mononuclear cells for CD34+ cells, without expanding cell number in vitro, so that at least 70% of the cells comprise CD34+ cells;
(c) at least 24 hours after step (a), passing a first portion of the enriched population produced in (b) through a catheter and confirming by testing in vitro that:
(1) at least some of the cells passed through the catheter have CXCR-4-mediated chemotactic activity and move in response to SDF-1 in vitro;
(2) at least 70% of the cells passed through the catheter are viable; and
(3) at least some of the cells passed through the catheter can form hematopoietic colonies in vitro;
(d) formulating for parenteral delivery through the catheter a sterile pharmaceutical composition comprising:
(i) a therapeutic amount of a sterile isolated chemotactic hematopoietic stem cell product,
wherein the therapeutic amount of the sterile isolated chemotactic hematopoietic stem cell product comprises a second portion of the enriched population produced in step (b) comprising at least 0.5×106 potent CD34+ cells expressing CXCR-4 that have CXCR-4 mediated chemotactic activity and that move in response to SDF-1 and is effective (i) to reduce cardiomyocyte cell death by improving perfusion (ii) to reduce cardiomyocyte apoptosis; and (iii) to preserve existing cardiomyocytes and cardiomyocyte function in the infarct area;
(ii) a stabilizing amount of serum, which is effective to retain the CXCR-4 mediated chemotactic activity and hematopoietic colony forming activity of the subpopulation of CD34+/CXCR-4+ cells that move in response to SDF-1 and
(iii) a therapeutic amount of at least one compatible therapeutic agent selected from the group consisting of a diuretic, an anti-arrhythmic agent, an anti-anginal agent, a cytokine, a tyrosine kinase receptor agonist, a vasoactive agent, an anticoagulant agent, a fibrinolytic agent, a hypercholesterolemic agent, or a combination thereof, which is effective to promote function of the existing cardiomyocytes to compensate for loss of cardiomyocyte function due to cardiomyocyte death; and
(e) administering to the subject parenterally through the catheter to treat the adverse ventricular remodeling at one or more infusion dates the sterile pharmaceutical composition.

2. The method according to claim 1, wherein the therapeutic amount of the chemotactic hematopoietic stem cell product comprises at least 10×106 isolated autologous CD34+ cells which further contain the subpopulation of at least 0.5×106 potent CD34+/CXCR-4+ cells that have CXCR-4 mediated chemotactic activity and that move in response to SDF-1.

3. The method according to claim 1, wherein the infarct area injury comprises apoptotic cardiomyocyte loss in the infarct area, a progressive decline in heart muscle function following the acute myocardial infarction, hypoperfusion of at least one ischemic peri-infarct zone of myocardial tissue, myocardial hibernation in the peri-infarct border zone, or a combination thereof.

4. The method according to claim 1, further comprising dividing the nonexpanded, isolated population of autologous mononuclear cells acquired in (a) into a plurality of aliquots; and freezing at least one aliquot at −86 degrees Celsius.

5. The method according to claim 1, wherein for at least 48 hours following the acquiring of the isolated population of autologous mononuclear cells in (a), when tested in vitro after passage through a catheter:
(a) the cells of the composition are capable of forming hematopoietic colonies in vitro; and
(b) the composition retains at least 2% of the CXCR-4-mediated chemotactic activity of the subpopulation of potent CD34+/CXCR-4+ cells that have CXCR-4-mediated chemotactic activity and that move in response to SDF-1.

6. The method according to claim 1, wherein for at least 72 hours following the acquiring of the isolated population of autologous mononuclear cells in (a), when tested in vitro after passage through a catheter:
(a) the cells of the composition are capable of forming hematopoietic colonies in vitro; and
(b) the composition retains at least 2% of the CXCR-4-mediated chemotactic activity of the subpopulation of potent CD34+/CXCR-4+ cells that have CXCR-4-mediated chemotactic activity that move in response to SDF-1.

7. The method according to claim 1, wherein for at least 24 hours following the acquiring of the nonexpanded, isolated population of autologous mononuclear cells in (a), when tested in vitro after passage through a catheter:
the composition retains at least 2% of the CXCR-4-mediated chemotactic activity of the subpopulation of potent CD34+/CXCR-4+ cells that have CXCR-4-mediated chemotactic activity.

8. The method according to claim 1, wherein the administering is through the catheter intravascularly to an infarct related artery.

9. The method according to claim 1, wherein the administering is through the catheter into myocardium.

10. The method according to claim 1, wherein the catheter is a flow control catheter.

11. The method according to claim 1, wherein the catheter is a balloon dilatation catheter.

12. The method according to claim 1, wherein the catheter has an internal diameter of at least about 0.36 mm.

13. The method according to claim 1, wherein the tyrosine kinase receptor agonist is neuregulin 1.

14. The method according to claim 1, wherein the sterile pharmaceutical composition of step (c) is more effective in treating adverse remodeling than a composition consisting of the therapeutic amount of the sterile isolated chemotactic hematopoietic stem cell product and the stabilizing amount of serum or a composition consisting of the therapeutically effective amount of at least one compatible therapeutic agent selected from the group consisting of a diuretic, an anti-arrhythmic agent, an anti-anginal agent, a cytokine, a tyrosine kinase receptor agonist, a vasoactive agent, an anticoagulant agent, a fibrinolytic agent, a hypercholesterolemic agent, or a combination thereof.

15. The method according to claim 1, wherein the method improves microvascular blood flow in the infarct area, decreases area of the infarct injury, decreases infarct mass, increases perfusion of at least one ischemic peri-infarct zone of myocardial tissue, increases perfusion to hibernating myocardium in at least one peri-infarct zone of myocardial tissue, or a combination thereof, when compared to controls.

16. The method-according to claim 1, wherein the cytokine comprises a vascular endothelial growth factor selected from the group consisting of VEGF-A, VEGF-B, VEGF-C, and VEGF-D.

17. The method according to claim 1, wherein the compatible therapeutic agent comprises placental growth factor.

18. The method according to claim 1, wherein the compatible therapeutic agent comprises a catecholamine.

19. The method according to claim 18, wherein the catecholamine is norepinephrine.

20. The method according to claim 1, wherein the compatible therapeutic comprises endothelin-1.

21. The method according to claim 1, wherein the compatible therapeutic comprises prostaglandin F2α.

22. The method according to claim 1, wherein the compatible therapeutic comprises angiotensin II.

23. The method according to claim 1, wherein the compatible therapeutic comprises phorbol ester.

24. The method according to claim 1, wherein the compatible therapeutic comprises neuropeptide Y.

25. The method according to claim 1, wherein the compatible therapeutic comprises active transforming growth factor β1.

26. The method according to claim 1, wherein the compatible therapeutic comprises Gq protein.

27. The method according to claim 1, wherein the compatible therapeutic comprises diacyl glycerol.

28. The method according to claim 1, wherein the compatible therapeutic comprises salusin-α.

29. The method according to claim 1, wherein the compatible therapeutic comprises salusin-β.

30. The method according to claim 1, wherein the compatible therapeutic comprises insulin-like growth factor-1.

31. The method according to claim 1, wherein the compatible therapeutic comprises myostatin.

32. The method according to claim 1, wherein the compatible therapeutic comprise granulocyte colony-stimulating factor.

33. The method according to claim 1, wherein the compatible therapeutic comprises macrophage colony-stimulating factor.

34. The method according to claim 1, wherein the compatible therapeutic comprises thiazolidinedione.

35. The method according to claim 34, wherein the thiazolidinedione is rosiglitazone.

36. The method according to claim 1, wherein the compatible therapeutic comprises necrosis factor-like weak inducer of apoptosis.

37. The method according to claim 1, wherein the cytokine is at least one selected from the group consisting of vascular endothelial growth factor (VEGF), placental growth factor (PIGF), granulocyte colony-stimulating factor (G-CSF), and macrophage colony-stimulating factor (M-CSF).

38. The method according to claim 1, wherein the subject is a revascularized subject.

39. The method according to claim 1, wherein administering the sterile pharmaceutical composition at one or more infusion dates is effective to maintain adequate perfusion, to support cardiomyocytes in a peri-infarct border zone and to preserve or restore viability of periinfarct myocardium.

40. The method according to claim 1, wherein the vasoactive agent is an angiotensin converting enzyme inhibitor.

41. The method according to claim 1, wherein the anti-arrhythmic agent is a beta blocker.